Proliferative responses of normal rat ventral prostate in vitro were investigated using the incorporation of 5-[125I]-iodo-2'-deoxyuridine (125I-UdR) to monitor DNA synthesis, with the aim of establishing a model system for evaluating the direct effects of hormones and chemotherapeutic agents on prostatic growth. In androgen-free, chemically-defined organ culture, the proliferative activity of young adult rat (4 to 6 months old) ventral prostate declined with time and the tissue underwent retrogressive changes resembling post-castration atrophy in vivo. Treatment with testosterone (4 x 10-12 to 4 x 10-5M) exhibited a dose-dependent response, with concentrations ranging from 4 x 10-9to 4 x 10-6M preventing the retrogressive changes associated with androgen deprivation and eliciting maximal increases (approximately 3-fold) in 125I-Udr uptake. Higher testosterone concentrations (2.5 x 10-5 to 4 x 10-5M) exerted a non-specific cytotoxic effect, resulting in marked suppression of 125I-Udr uptake. Following stimulation with an optimal concentration of testosterone (4 x 10-7M), 125I-Udr incorporation reached peak activity on day 4 of the culture period and rapidly declined thereafter, despite the continued presence of testosterone. Effects of variations in organ culture media and methodology were also examined to further establish optimal conditions for investigations of the proliferative response to testosterone stimulation. The established method of quantitative organ culture was then used to compare the proliferative effect of testosterone with that of its major metabolites, 5α-dihydrotestosterone, androstenedione, androstenedione and 5α-androstane-3β, 17β-diol.