The human immunodeficiency virus rev gene product regulates the expression of viral structural genes. It was recently shown that Rev regulates the export of viral structural mRNAs from the nucleus to the cytoplasm. Analysis of Rev subcellular localization reveals marked accumulation in the nucleolus, suggesting a role for the nucleolus in this export process. We report here the identification of amino acid residues critical to the nucleolar localization of Rev. Consistent with this finding, a Rev/beta-galactosidase fusion protein, harboring this region of Rev, localized entirely within the nucleolus. Of most significance, mutations that eliminated nucleolar localization markedly diminished Rev function, even though accumulation in the nucleoplasm was retained. These findings support a model whereby Rev-induced export of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm is likely to involve nucleolar events.