A full-length clone encoding cytochrome b5 has been isolated from a tobacco leaf cDNA library in lambda gt11 by PCR using degenerate primers. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochrome b5s, the message for which is expressed predominantly in developing seeds and in pigmented flower tissue. In the developing tobacco seed the mRNA is abundant at very early stages (< 10 days after flowering). Southern analysis indicated that more than one gene encodes cytochrome b5 in the tobacco genome. In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathway and that the C-terminus of the protein is required for targeting and insertion.