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 K_cat/K_m 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.