Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group.