Administration of androgen to the castrate rat elicits a pronounced wave of proliferative activity in the ventral prostate gland. We wished to determine if this phenomenon could also be demonstrated in malignant prostate tissue. An experimental protocol consisting of 12 days of androgen depletion induced by castration followed by 7 days of androgen repletion was utilized in animals bearing the androgen-dependent Dunning R3327H prostatic adenocarcinoma. Repletion of androgen levels was achieved by daily s.c. injection of testosterone or dihydrotestosterone at 8, 16, or 32 mg/kg of animal body weight. The proliferative response of the tumor to conditions of depletion as well as repletion on Days 3, 5, and 7 was determined using [3H] thymidine autoradiography, quantitative morphometry, and flow cytometric analysis. The autoradiographic and flow cytometric data were complementary and indicated that androgen depletion caused a slight reduction in the percentage of S-phase nuclei. Repletion initiated a highly reproducible, significant increase in both the S-phase compartment as well as tritiated thymidine incorporation into DNA, and of the days quantitated, the greatest values were obtained on Day 3. The response patterns were nearly identical for both testosterone and dihydrotestosterone, and no significant differences were detected among the doses used. Quantitation of the autoradiographs revealed a striking disparity in the response of the cell types. The labeling indices of nonepithelial cells increased only minimally during repletion, whereas the epithelial cells responded consistently and reached levels 2- to 4-fold over intact values. These data indicate that protocols of androgen depletion/repletion have the capacity to elicit a significant wave of cell proliferation. These manipulations support the feasibility of transiently increasing the number of cancer cells in S phase as a means of potentiating cytotoxic chemotherapy for treatment of adenocarcinoma of the prostate.