The prostate gland is unusual in that it has a high content of citric acid compared with other soft tissues. The mechanism of citrate accumulation in prostate is unknown. One possibility is that aspartate aminotransferase (AAT) could supply an extra Krebs cycle source of oxaloacetate for citrate formation. Since testosterone regulates citrate production and secretion and strong transaminase activity exists in prostate, a relationship between AAT and citrate production could exist if testosterone also regulates AAT activity. To investigate this possibility, animals were castrated and given testosterone. After a total castration period of 72 hours, animals were sacrificed and mitochondrial AAT activity determined. Results revealed that castration decreased AAT activity and testosterone replacement stimulated activity. Neither castration nor testosterone affected malate dehydrogenase (MDH) activity. Both actinomycin D and cycloheximide blocked the testosterone stimulation of AAT activity. These studies indicate that testosterone may regulate AAT activity in rat ventral prostate. Furthermore, regulation may occur through testosterone stimulation of specific RNA and protein synthesis.