alpha-Naphthoflavone (ANF; 7,8-benzoflavone) is a potent competitive inhibitor of human aromatase cytochrome P-450 [J. T. Kellis, Jr. and L. E. Vickery, Science 225, 1032 (1984)]. We have further investigated inhibition of aromatase by several derivatives of ANF. Using human placental microsomes and 40 nM androstenedione as substrate, the compounds tested and their I50 values were: ANF, 0.07 microM; 2-(2-naphthyl)-4H-naphtho[1,2b]pyran-4-one, 1.0 microM; 7,8-benzoisoflavone, approximately 100 microM; and 2-phenyl-4H-naphtho[1,2b]furan, greater than 100 microM. These findings show the necessity of the keto group of ANF in its binding to the enzyme and the importance of size and position of substitution of the exocyclic phenyl ring. Derivatives of ANF with hydroxyl substitution at positions 5, 6, 7, 8, 9, and 10 were also screened. 9-Hydroxy-ANF, a known metabolite of ANF in liver microsomes, was the most effective (I50 = 20 nM). Inhibition by 9-hydroxy-ANF was competitive, and its Ki value of 5 nM indicates a higher affinity for the enzyme than the natural steroid substrates--the Km values for androstenedione and testosterone under these conditions are 10 and 80 nM respectively. 9-Hydroxy-ANF also induced a change in the absorption spectrum of hte aromatase cytochrome P-450 indicative of substrate displacement. Based on these data we propose a model for the binding of 9-hydroxy-ANF in which the 7,8-benzochromone ring system of the ANF derivatives occupies the steroid ring binding site of the enzyme.