The fibromuscular stroma (FMS) separated from the seminal vesicles of adult guinea pigs incorporated [35S]methionine into a 12 500 X g soluble fraction in vitro. Incorporation decreased 60% in tissues from 7-day castrates with no further change up to 21 days post-castration. Administration of 5 alpha-dihydrotestosterone prevented this decrease. Castration did not appear to increase the degradation of newly synthesized protein, or to alter the distribution between soluble and particulate subcellular fractions. Using SDS-PAGE and fluorography to quantitate [35S]methionine incorporation into specific proteins, three protein classes were identified which responded differentially to castration: (1) bands at 110 000 and 24 000 Mr decreased by 95%; (2) bands at 58 000 and 97 000 Mr were unchanged; (3) multiple bands decreased in proportion to total incorporation. Administration of 5 alpha-dihydrotestosterone could not fully maintain, at control levels, the synthesis of the 110 000 and 24 000 Mr bands. These results demonstrate that the FMS of the guinea pig seminal vesicle can serve as a useful model system for studying hormonally regulated functions of male sex accessory stromal tissue.