Dihydrotestosterone (DHT) is the active androgen, as well as a strong tumor promoter in the prostate, where several enzymes are essential for the regulation of its activity. We localized four enzymes promoting the enolization of the 3-keto group of DHT in rat prostate. The enzymes were purified by ion-exchange chromatography, acetone fractionation and gel filtration to homogeneity, and found to have molecular sizes of 19.5, 22.0, 44.5 and 21.5 kDa. A partial characterization of the four enzymes revealed that their structure consisted of a common chain of 14.5 kDa with various subunits which differentiate the four enzymes from each other. All the enzymes exerted their activity only on 5-dihydro 3-keto steroids. The total enzymatic activity was strongly influenced by animal age, being very low before sexual maturation, as well as after castration. In the latter case the level of total activity fell to about 8% of control animals. Activity was also estimated in human, pork, ram and bovine prostate and it was found that all these species have 20 - 25 times lower enzyme levels than rat. These results, in combination with the practically exclusive localization of the enzymes in the prostate, suggest a role relating to the bioavailability of DHT in this gland