The ability of mammary tissue from mature Holstein bulls to respond to hormonal stimuli in organ culture to synthesize and secrete milk proteins was studied. Sixteen bulls from the genetic selection and control lines developed at Virginia Polyteclmic Institute were randomly assigned to either a non-treated or steroid pretreated group. The steroid pretreated group received estradiol 17-B and progesterone at 0.1 and 0.25 mg/kg/day, respectively, for 7 d starting 15 d before slaughter. Mammary tissue was explanted and cultured for up to 96 h in basal medium (B) which consisted of Media 199 supplemented with fetal calf serum and bovine insulin, or stimulatory medium (St), which was further supplemented with triiodothyronine, estradiol 17-B, hydrocortisone, and bovine prolactin (PRL). Explants in duplicate multi-well culture dishes were incubated in the presence of 3H-Iabeled amino acids or 3H-Iabeled thymidine. Measurements made included: casein in media and homogenized tissue, alpha-lactalbumin in homogenized tissue, tri-chloro-acetic acid precipitable 3H-protein in media and homogenized tissue, and DNA content of cultured tissue. A subset of cultured explants from each bull was fixed and embedded for histological evaluation and auto radiographic localization of incorporated 3H-labeled thymidine. Synthesis and secretion data, as well as growth responses of the epithelium were compared between selection and control bulls.