The amino acid sequence of LepA protein, which has been shown to be cotranscribed with signal peptidase I in Escherichia coli, was compared with greater than 2000 known protein sequences. It was revealed that, of the 598 amino acid residues contained in LepA, an amino-terminal domain of 112 residues is homologous to a domain of similar size found in initiation factor 2, elongation factor Tu, and elongation factor G (IF2, EF-Tu, and EF-G), factors required for translation in E. coli. In this domain, 46 and 34 residues align perfectly with the corresponding regions of EF-G and EF-Tu, respectively. If functionally conserved residues within this domain (19 for EF-G and 17 for EF-Tu) are included, the overall resemblance is 58% and 46%, respectively, for EF-G and EF-Tu. A similar domain exists internally in IF2, where there is 42% overall resemblance with the domain of LepA. Immediately adjacent to this region is a small sequence of limited similarity that exists not only in EF-G, EF-Tu, and IF2 but also in the protooncogene c-Ha-ras-1 (from human bladder) and other GTP-binding proteins. Given these homologies, GTP-photoaffinity labeling and subcellular fractionation experiments were undertaken, and it was found that LepA is indeed a membrane-bound GTP-binding protein.