Interactions of the substrate(s) benzphetamine and the product (P) desmethylbenzphetamine with cytochrome P450 2B4 were studied by difference spectrophotometry. A two-sites model in which site 1 binding, causing Type I transition (low- to high-spin) must precede site 2 binding, causing Type II transition (high- to low-spin), gave an acceptable fit to the spectral titration data. The equilibrium association constant of substrate for site 1 (K1) was greater than that for site 2 (K2), and the K2 for the product was greater than K1, indicating that the substrate binds preferentially to site 1 and the product prefers site 2. In addition, competition between P and a strong Type II ligand (1-benzylimidazole) and a noncompetitive type of interaction between S and the same Type II ligand was observed. This indicates that P binds to the same site as the Type II ligand and S binds to a different site. The observed high-spin maxima for both P (EP1HSmax) and S (ES1HSmax) were similar to those calculated using the K1 and K2 values obtained from the curve-fitting procedure, indicating that the equilibrium concentration of the high-spin species is controlled entirely by K1 and K2. Simultaneous presence of the substrate and product decreased K1 of the substrate and K2 of the product, indicating that there is interaction between the substrate-preferred and the product-preferred sites. A possible functional significance of the differences in the site preferences of the substrate and product is discussed.