CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5α-androstane-3β,17β-diol (3β-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3β-Adiol may play an important role for estrogen receptor β-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor β activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3β-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3β-Adiol (a CYP7B1 substrate) and 7α-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor β activation. The data obtained indicated that 3β-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor β activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3β-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the Kcat/Km values were in the same order of magnitude. A high dehydroepiandrosterone/3β-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3β-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3β-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3β-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor β signaling.