We examined in vitro translocation of pro-OmpA derivatives with a His6 tag at various positions in their mature proteins and with a c-Myc tag at their C termini across inverted membrane vesicles of Escherichia coli. Those with a His6 tag in the N-terminal region of the mature domain, which corresponds to the "translocation initiation domain" proposed previously (Andersson, H., and von Heijne, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9751-9754), could not be translocated in the presence of 100 micron Ni2+, while OmpA derivatives with a His6 tag in the middle of or at the C terminus did not show such Ni2+ sensitivity. The inhibitory action of Ni2+ on pro-3His-OmpA' (with a His6 tag after the third amino acid of the mature OmpA-c-Myc region) translocation was exerted only during early events, after which it became ineffective. The inhibition point of Ni2+ was suggested to lie between membrane targeting and exposure of the signal cleavage site to the periplasm since the unprocessed and membrane-bound form of pro-3His-OmpA' was accumulated by the addition of Ni2+. The Ni(2+)-"trapped" precursor was released from its translocation block by 30 mM histidine, which should compete with the His6 tag on the precursor protein for formation of a Ni2+ chelating complex. We propose that Ni2+ confers a reversible positive charge effect on the His6-tagged initiation domain of the pro-OmpA derivatives and inhibits an early event(s) of protein translocation, such as presentation of the precursor to the membranous part of the translocase. This system will be useful in dissecting early events of the protein translocation pathway.