Experiments were conducted to investigate: 1) production of trans-18:1 and cis/trans-18:2 isomers due to input of forage and corn grain in continuous culture fermenters, 2) concentrations of trans-18:1 and cis/trans-18:2 isomers in blood and milk fat of grazing cows fed a grain supplement containing solvent- or mechanically-extracted soybean meal, 3) plasma and milk fatty acid profiles of lactating cows in response to a conjugated linoleic acid (CLA) mixture infused into the rumen, and 4) effects of cis9,trans11-18:2 (9/11CLA) or trans10,cis12-18:2 (10/12CLA) on de novo synthesis and desaturation of milk fatty acids in lactating cows fed unsaturated oils. In the first study, rumen fermenters were fed (50 g DM/d) orchardgrass or red clover with 0, 8, or 16 g/d corn replacing equal portions of forage DM. Outflow of trans11,cis15-18:2 (11/15LA) in effluents was greater when DM input was clover or grass only. With each increment of corn, output of 9/11CLA increased until it was 205% greater compared with forage alone. Trans11-18:1 (TVA), an intermediate in 18:2n6 (LA) and 18:3n3 (LN) hydrogenation, output increased by 28% with corn addition. Outputs of trans10-18:1 and 10/12CLA nearly doubled as corn increment increased. In the second experiment, Holstein cows grazing mixed clover-grass pastures were fed a grain supplement (6.7 kg/d) containing 1.7 kg solvent-extracted soybean meal (SES, 15 mg LA/g of DM), 1.9 kg mechanically-extracted soybean meal (MES, 24 mg LA/g of DM), or 1.9 kg MES plus 30 g of liquid methionine hydroxy analog (MESM). Cows fed MES or MESM had greater concentrations of LA, TVA, 9/11CLA, and 11/15LA in blood compared with cows fed SES. Daily yields of 18:0 (SA), LA, LN, TVA, and 9/11CLA in milk fat also were greater for cows fed MES or MESM compared with SES. In experiment 3, four Holstein cows were used in a 4 x 4 Latin square to determine plasma and milk fatty acid profiles during infusion of a CLA mixture at 0, 45, 90, or 180 g/d for 48 h into the rumen. Relative to the control, infusion of 180 g CLA/d decreased milk fat percentage and yield. Lower milk fat yield resulted from depressed concentrations of saturated 6:0 to 16:0 medium-chain fatty acids (MCFA). Concentrations of TVA, trans10-18:1, and 10/12CLA in blood plasma, and yields in milk fat increased in response to each dose of CLA. Stearic acid yield also increased as dose of CLA increased. Yield of cis9-18:1 (OA) in milk fat, however, was lower at 180 g CLA/d. In experiment 4, four cows were fed high-oleic (HO) or high-linoleic (HL) (2.5% of DM) oil for 11 d prior to abomasal infusion (15 g/d) of 9/11CLA or 10/12CLA for 48 h (2 x 2 factorial). Milk fat percentage and yield decreased 25% due to infusion of 10/12CLA compared with 9/11CLA, regardless of diet. Lower fat yields resulted from lower MCFA concentrations and yields. Regardless of diet, concentration (but not yield) of SA increased 40% when 10/12CLA was infused compared with 9/11CLA. Concentrations and yields of OA, 9/11CLA, and 20:4n6 also were reduced by infusing 10/12CLA compared with 9/11CLA regardless of diet. Thus, in addition to inhibiting de novo fatty acid synthesis, 10/12CLA appeared to inhibit desaturation via delta-6 and delta-9 desaturases. Significant implications from the above studies include: 1) 11/15LA and TVA are the primary intermediates flowing out of the rumen during hydrogenation of pasture lipids, 2) replacing forage DM with starch, OA, and LA increases synthesis of trans10-18:1 and 10/12CLA in the rumen, 3) desaturation of TVA produced in the rumen provides an alternate source for 9/11CLA in milk fat, and 4) 10/12CLA decreases de novo synthesis and desaturation of milk fatty acids.