Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5 ) in some but not all microsomal cytochrome P450 (P450, CYP) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5 , that could be utilized in binding assays at sub-µM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1 with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5 -POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high and that ternary P450-b 5 -POR complexes are relevant in P450 3A4 reactions, as opposed to a shuttle mechanism.