Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, alpha-naphthoflavone (ANF) and the steroid, testosterone (TST). UV-vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: (1) a relatively tight spin-state insensitive binding site (CYP.ANF) and (2) a relatively low affinity spin-state sensitive binding site (CYP.ANF.ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP.ANF complex is strongly temperature dependent, when compared to the formation of the CYP.TST complex, suggesting that the formation of the CYP3A4.ANF complex leads to long-range conformational changes within the protein. When the CYP.ANF complex is titrated with TST, the formation of CYP.ANF.TST is favored by 3:1 over the formation of CYP.TST.TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST.