Evidence for the inhibitory effect of dehydroepiandrosterone (DHEA) on the in vitro cleavage of dehydroepiandrosterone sulfate (DHEA-SO4) by rat testis tissue was obtained. Whole homogenates of rat testis tissue were incubated with (1) DHEA-7α-3H with varying amounts of DHEA-SO4-NH4, (2) DHEA-7α-3H-SO4 with varying amounts of DHEA, (3) DHEA-7α-3H-SO4 with varying amounts of DHEA-4-14C, and (4) DHEA-7α-3H-SO4 with 100 μg of testosterone-4-14C. Time points of 5, 15, and 60 min were studied. Labeled DHEA was converted to steroid sulfates in amounts (5–10%) not affected by the amounts of DHEA-SO4 present. The amount (7–11%) of DHEA-7α-3H-SO4 converted to unconjugated steroids was decreased in the presence of either exogenous DHEA or testosterone. The distribution of label in the unconjugated steroids arising from DHEA-7α-3H-SO4 was similar to the distribution observed from labeled DHEA.