Cytochrome P450-dependent drug metabolism in vitro frequently deviates from simple Michaelis-Menten kinetic models, and demonstrates both positive and negative homotropic and heterotropic effects. These complex "allsoteric" kinetics confound our ability to predict drug clearance, and they may provide a basis for drug-drug interactions. Although allosteric effects require that multiple substrates, or substrate and effector, are simultaneously bound to a cytochrome P450 (CYP), the mechanisms by which multiple ligand binding alters rates of individual steps in the CYP reaction cycle are incompletely characterized. In addition, it is unknown whether multiple ligands bind in discrete subsites within the large active site or whether they share a fluid dynamic site. These mechanistic aspects of multiple drug binding are addressed here via several spectroscopic probes including ultraviolet-vis difference spectroscopy, protein and ligand fluorescence, and 15N-edited HSQC nuclear magnetic resonance (NMR) with 15N-Phe-labeled CYPs. The results indicate a lack of correspondence between ligand binding per se and the ligand-dependent home spin state change when multiple ligands bind. Furthermore, the results provide proof of principle for NMR as a method for studying CYP allosterism, and demonstrate that the model ligand 9-aminophenanthrene binds in two discrete events to individual subsites within the active site of CYP(eryF).