The HIV-2 genome contains an open reading frame (designated X-orf) that does not have a counterpart in HIV-1. To establish whether X-orf is a gene, we studied its expression in HIV-2-infected individuals and in infected cells in vitro. An HIV-2 proviral DNA fragment containing the X-orf was expressed in E. coli, and the recombinant protein was used in an immunoblot assay. The X-orf protein was recognized specifically by the sera of HIV-2-infected people but not by the sera of SIV-infected monkeys or HIV-1-infected humans. A rabbit antiserum raised against the recombinant X-orf protein recognized a 16 kD protein in HIV-2-infected cells. The native X-orf protein was not glycosylated or phosphorylated, was localized in the cytoplasm of HIV-2-infected cells, and appeared to be associated with mature virions.