Mammary gland samples from a large serial-slaughter Holstein heifer nutrition trial were used to determine the effects of stage of development and nutritional management on mitogenic activity of mammary extracts and mammary parenchymal composition. Stage of development and nutritional management of heifers had minimal effects on the mitogenic capacity of mammary extracts, and no significant effects on tissue composition. Two-dimensional proteome maps of heifer mammary extracts were constructed for heifers weighing 200 and 350 kg, respectively at slaughter. Proteins altered by stage of development and/or nutrition were quantified and identified; 820 total protein spots were analyzed. The expression of 131 protein spots differed by dietary treatment only. Stage of development altered the expression of 108 protein spots. Twenty-two protein spots were excised from gels for mass spectrometry analyses. Database searches for proteins with shared primary amino acid sequences were used to identify the proteins. Possible roles of these proteins in mammary development were described. In summary, heifers can be reared on high planes of nutrition without impairing mammary development, but mechanisms governing nutritional and temporal control of mammary development demand further investigation.