Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b5 indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b5, including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b5 in the endoplasmic reticulum membrane. Precytochrome b5 was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b5 was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b5 in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b5 with microsomes resulted in a “loose” incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b5 has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b5 to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.