The membrane-bound protein Cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high-density lipoprotein particles, whose stability, monodispersity and consistency are ensured by their self-assembly. We explore the energetics of four ligands: (6-(p-toluidino)-2-naphthalenesulfonic acid, TNS; a-naphthoflavone, ANF; miconazole; and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the Nanodiscs’ scaffold proteins; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 µM for TNS, 5.8 µM for ANF, 0.45 µM for miconazole and 0.45 µM for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer, and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment, ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site