We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H2O release method as described by Weisz. Production of 3H2O from 1 beta-[3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein X h in 3-month old rats to 68 pmol/mg protein X h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.