A model previously developed in our laboratory to culture rat prostate explants in serum-free chemically-defined medium was used to evaluate the direct influence of potential regulators. The aim of the present work was to verify the effects of insulin (I) and transferrin (Tr), two hormones considered as essential in other serum-free culture systems, and three androgenic hormones, since the prostate is known to be androgen-dependent. Explants of rat prostate were cultured for five days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air-5% CO2). The addition of Tr (50 micrograms/ml) had no effect, but I (5 micrograms/ml) significantly increased DNA synthesis. This influence was amplified by combination of the two hormones. However, protein synthesis was only slightly stimulated. Testosterone (T) or androstanediol significantly increased DNA synthesis when compared to corresponding control values at five days. In combination with I plus Tr, each hormone showed potentiated effects, particularly T with a twofold increase over day 0 values. When dihydrotestosterone was added singly, the incorporation of 3H-thymidine was stimulated by 300% over control values at five days, and by 100% over values in uncultured explants. This influence was maximal since it was not improved by I plus Tr. Protein synthesis was increased significantly by the triple combination. In addition, each androgen as well as the combination of I plus Tr had a positive influence on explant morphology. The above conditions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors in prostate metabolism and to eventually identify putative cancer markers.