We have studied the membrane topology of liver microsomal cytochromes P-450 derived from phenobarbital-treated rabbits via trypsinolysis of intact microsomes, recovery of solubilized peptide fragments by ultracentrifugation and liquid chromatography, primary structure determination by Edman microsequence analysis, and database searching to match isolated fragments with parent sequences. Relative to the primary structure of isozyme 2, the major phenobarbital-inducible form, fragments were isolated beginning at residues Glu86, Ile101, Arg126, Cys152, Leu198, Ser211, Asn237, Glu327, Asn385, Thr407, Phe408, Phe413, and Thr444. Such results show that this family of structurally related cytochromes is bound to the endoplasmic reticulum membrane by only one or two transmembrane segments, located at the NH2-terminal end of the polypeptide chain. The remainder of the protein, from residue approximately 50 to the COOH terminus must exist as a catalytic, heme-containing domain exposed on the cytosolic side of the membrane. Furthermore, our results indicate that the catalytic domain must be peripherally associated with the membrane surface. This would imply that substrates might have access to the active site of the cytochrome P-450 either by diffusion from the cytosol or from within the lipid bilayer.