In order to investigate the role of protein synthesis in the testosterone regulation of androgen receptor (AR) levels, in vivo studies were undertaken using the ventral prostate gland from adult male rats castrated 24 h previously. Our results showed that testosterone (400 microg/100 g body weight) increased nuclear AR binding 1 h after administration, whereas the protein synthesis inhibitor cycloheximide (400 microg/100 g body weight) by itself did not alter AR binding. However, concomitant administration of testosterone and cycloheximide blocked the testosterone-induced nuclear AR accumulation after 1 h. To determine if changes in AR binding reflected changes in AR protein levels, immunocytochemical studies were conducted on individually dissected ventral prostatic ducts. Castration 24 h previously induced a decrease in nuclear AR immunostaining when compared to intact animals. Testosterone treatment restored the nuclear staining, particularly at the distal tips of the prostatic ducts. Cycloheximide alone did not change AR immunostaining when compared to castrated vehicle-treated rats, yet it significantly decreased the nuclear AR staining induced by testosterone. Our results suggest that AR is being newly synthesized during testosterone treatment. To determine if the effect of testosterone in the regulation of the AR protein was ultimately due to changes at the messenger RNA (mRNA) levels, steady-state AR mRNA levels were measured. Northern blot analysis of poly A+ mRNA preparations revealed that androgen withdrawal for 24 h increased AR mRNA and that testosterone treatment for 1 h did not alter these increased AR mRNA levels. The inhibition of protein synthesis by cycloheximide did not change AR mRNA, but, when cycloheximide was administered in conjunction with testosterone, AR mRNA levels were significantly decreased. In an attempt to relate these responses to changes in transcriptional activity, the transcription inhibitor actinomycin D was administered in vivo. Whereas simultaneous administration of testosterone and cycloheximide modified AR mRNA and AR protein levels, concomitant administration of testosterone with actinomycin D did not alter these levels. It is therefore unlikely that testosterone modifies the transcription of AR mRNA within 1 h after its administration. Collectively, these results suggest that protein synthesis is involved in the mechanism of testosterone-promoted AR regulation. This protein synthesis-dependent mechanism may be involved in the regulation of the stability and/or the translation of AR mRNA in the prostate.