Based on data indicating that compounds which induce cytochrome P-450d also bind to the enzyme [R. Voorman and S. D. Aust (1987). Toxicol. Appl. Pharmacol. 90, 69-78], we investigated the inhibition of cytochrome P-450d-dependent estradiol 2-hydroxylase by (2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD), using ligand-free cytochrome P-450d from isosafrole-treated rats. Since maximum inhibition of estradiol 2-hydroxylase occurred at TCDD concentrations comparable to the concentration of enzyme (50 nM), a modified form of steady-state kinetic analysis was used. Using I50 = Et/2 + Ki where Et = total enzyme concentration), we showed that TCDD inhibited cytochrome P-450d-dependent estradiol 2-hydroxylase activity with Ki equal to 8 nM. Association of TCDD with P-450d occurred within 2 min of inhibitor addition. Therefore, TCDD can be considered a tight binding inhibitor of cytochrome P-450d.