The membrane topology of cytochrome P450 2B4 from the endoplasmic reticulum has been studied with highly-purified liver microsomes in a site-directed immunochemical approach. Microsomes were prepared from phenobarbital-induced rabbits, and the resulting microsomal fraction was washed 6 additional times with 0.1 M pyrophosphate buffer to effect removal of significant quantities of adventitiously-bound protein. Monoclonal antibodies were prepared against residues 18-29 of P450 2B4 (Leu18-Leu-Phe-Arg-Gly-His-Pro-Lys-Ala-His-Gly-Arg29), essentially corresponding to the halt-transfer signal. This region was chosen due to its mutually-exclusive location in the two alternative membrane topology models currently tenable [Black, S.D. (1992) FASEB J.6, 680-685]. Model "A" contains a single transmembrane anchor peptide with the amino terminus projecting into the lumen of the endoplasmic reticulum, while model "B" exhibits a hairpin loop of the first approximately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer; the halt-transfer signal peptide would be located at the cytosolic surface of the membrane in model "A" or as a loop on the lumenal side of the membrane in model "B". Nine antibodies, denoted as MmAbA, MmAbC, MmAbD, MmAbF, MmAbH, MmAbI, MmAbK, MmAbL, and MmAbP, were produced, and all were identified as IgM/kappa subtypes. Western blotting demonstrated that the antibodies could readily recognize P450 2B4 in microsomes. ELISA assays showed that all of the antibodies exhibited strong binding to intact microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)