Although responsible for a major health problem worldwide, hepatitis C virus is difficult to study because of the absence of fully permissive cell cultures or experimental animal models other than the chimpanzee. GB virus B (GBV-B), a closely related hepatotropic virus that infects small New World primates and replicates efficiently in primary hepatocyte cultures, is an attractive surrogate model system. However, little is known about processing of the GBV-B polyprotein. Because an understanding of these events is critical to further development of model GBV-B systems, we characterized signal peptidase processing of the polyprotein segment containing the putative structural proteins. We identified the exact N termini of the mature GBV-B envelope proteins, E1 and E2, and the first nonstructural protein, NS2, by direct amino acid sequencing. Interestingly, these studies document the existence of a previously unrecognized 13-kDa protein (p13) located between E2 and NS2 within the polyprotein. We compared the sequence of the p13 protein to that of hepatitis C virus p7, a small membrane-spanning protein with a similar location in the polyprotein and recently identified ion channel activity. The C-terminal half of p13 shows clear homology with p7, suggesting a common function, but the substantially larger size of p13, with 4 rather than 2 predicted transmembrane segments, indicates a different structural organization and/or additional functions. The identification of p13 in the GBV-B polyprotein provides strong support for the hypothesis that ion channel-forming proteins are essential for the life cycle of flaviviruses, possibly playing a role in virion morphogenesis and/or virus entry into cells.