Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques.