Canine prostatic epithelial cells were cultured in primary monolayers in order to define those factors that induce a proliferative response at the cellular level. Cultures were performed in a serum-free medium or in a medium supplemented either with fetal bovine serum or dog serum in the presence or absence of several sex steroids (androstenedione, testosterone, dihydrotestosterone, 3 alpha- and 3 beta-androstanediols, epitestosterone, epidihydrotestosterone, estrone, estradiol, and progesterone). Cell proliferation was observed in the absence of serum and exogenous steroids. The rate of cell division was serum dependent and steroid independent. Pretreatment of sera with charcoal had no effect on their mitogenic activities. Cells maintained in an endocrine milieu prior to tissue dispersion and throughout the whole procedure proliferate to the same extent as those deprived of hormones, whether free of serum or added supplements. The addition of insulin (2 micrograms/ml), dog prolactin (up to 25 ng/ml) and zinc (10(-8) to 10(-2) M) in a serum-free medium did not induce cell responsiveness to steroids. Dihydrotestosterone, 3 alpha-androstanediol, and estradiol alone or in combinations known to induce the growth of the canine prostate in vivo were ineffective in vitro. The proliferative responses to sera were time and concentration dependent, and dog serum was more potent than fetal bovine serum. Thus, humoral factors other than steroids, prolactin, insulin, or zinc may be of importance in the activation of epithelial cells involved in the development of prostatic hyperplasia and adenocarcinoma.