The interactions of lidocaine (LIDO) with two closely related P450s, 2B1 and 2B2, were investigated using chimeric enzymes and single-point mutants derived from the two proteins. P450 2B1 exclusively catalyzed the N-deethylation of LIDO to generate monoethylglycinexylidide (MEGX) and 2B2 catalyzed both N-deethylation and hydroxylation reactions to generate MEGX and omega-diethylamino-2-hydroxymethyl-6-methylacetanilide. The addition of LIDO to 2B2 evoked a type I binding spectral change with a measured Ks of approximately 20 microM. The magnitude of the change in the absorbance obtained following the binding of LIDO to 2B2 was indicative of an approximately 30% switch of the heme iron to the high-spin form. In contrast, the addition of LIDO to 2B1 resulted in less than a 1% shift to the high-spin form even at LIDO concentrations as high as 10 mM. P450 2B2 exhibited a low Km value for LIDO (62 microM), whereas 2B1 had an approximately 10-fold higher Km value. However, the rates of LIDO oxidation by 2B1 were approximately 200-fold those exhibited by 2B2. Substitution of 2B2 residues by 2B1-derived amino acids influenced the spectral binding, regioselectivity of LIDO oxidation, and the kinetic properties of the enzyme. With the 2B2 Ala-363 to Val mutant, a complete switch of the 2B2 mutant to catalyzing only the N-deethylation activity was observed. The altered regioselectivity was accompanied with approximately 10-fold increases in the measured Ks, Km, and kcat values for LIDO.