This study has identified the polar metabolites of 5 alpha-androstane-3 beta-17 beta-diol(3 beta-diol) produced by the canine prostate. The major metabolite is 5 alpha-androstane-3 beta,7 alpha,17 beta-triol(7 alpha-triol) accounting for approximately 80% of the total polar metabolites of 3 beta-diol. The remaining 20% is accounted for exclusively by another triol, 5 alpha-androstane-3 beta,6 alpha,17 beta-triol(6 alpha-triol). This study has also characterized two enzymatic hydroxylase responsible for respective triol formation: 5 alpha-androstane-3 beta,17 beta-diol 6 alpha-hydroxylase(6 alpha-hydroxylase) and 5 alpha-androstane-3 beta,17 beta-diol 7 alpha-hydroxylase(7 alpha-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several in vitro steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C19 androstane substrates, 6 alpha- and 7 alpha-triol were found to be major components of the total 3 beta-hydroxy-5 alpha-androstane metabolites produced by the canine prostate.