In order to examine the similarity of the protein translocation machineries of Escherichia coli and the marine bacterium Vibrio alginolyticus, a protein translocation system was reconstituted from inverted membrane vesicles prepared from V. alginolyticus, E. coli uncleavable OmpF-Lpp, and partially purified V. alginolyticus SecA. Translocation of OmpF-Lpp required ATP and was stimulated by an increase in the amount of V. alginolyticus SecA. In addition, translocation activity in the presence of E. coli SecA was strongly inhibited by a site-specific antibody raised against a synthetic peptide corresponding to the C-terminal region of E. coli SecY, suggesting that the C-terminus of V. alginolyticus SecY has homology with the C-terminus of E. coli SecY. Based on these findings, it is suggested that the protein translocation machinery of V. alginolyticus is similar to the E. coli Sec machinery.