Previously, we have proposed that inhibition of human placental steroid sulfatase by endogenous steroids may render it rate limiting and provide a mechanism controlling estrogen synthesis from C19-Δ5-3β-yl sulfates during pregnancy. Inhibitory effects of 129 endogenous and synthetic steroids on DHAS (l7-oxo-5-androsten- 3β-yl sulfate, apparent Km = 3.3 × 10-5M) hydrolysis by human term placental sulfatase have now been determined in vitro. Structure-activity data are compatible with binding of conjugated steroids to the enzyme via 3 sites, in addition to hydrophobic interactions. Inhibitory activity of unconjugated compounds is favored by planar Δ5-or 5α-structures unsubstituted except for oxygen functions at C-3 and C-20. Alternative substrates (e.g., pregnenolone sulfate, apparent Ki = 0.06 × 10-5M), acting competitively, were the most potent inhibitors. They appear to be the major potential factors reducing effective sulfatase levels in the proposed mechanism. Cumulative effects of other endogenous, competitive inhibitors (e.g., progesterone, Ki = 1.70 × 10-5M; DHA, Ki = 3.25 × 10-5M; estradiol-17β, Ki = 1.15 × 10-5M) may reduce sulfatase activity still further and contribute to the regulation of estrogen synthesis. Further studies using intact placental preparations and in vivo will be required to test this hypothesis. (Endocrinology93: 172, 1973)