The prolipoprotein, a secretory precursor of the outer membrane lipoprotein of Escherichia coli, is known to be accumulated in the cell envelope when cells are grown in the presence of a cyclic antibiotic, globomycin. The prolipoprotein was localized in the cytoplasmic membrane when it was separated from the outer membrane by sucrose-density gradient centrifugation. However, when the envelope fraction was treated with sodium sarcosinate, the prolipoprotein was found almost exclusively in the sarcosinate-insoluble outer membrane fraction. The prolipoprotein separated in the cytoplasmic membrane by sucrose-density gradient centrifugation was soluble in sarcosinate and could not form a complex with the outer membrane once solubilized in sarcosinate. Labeling of the two lysine residues at positions 2 and 5 of the prolipoprotein with [3H]dinitrophenylfluorobenzene was enhanced 26-fold when the cells were disrupted by sonication. On the other hand, a tryptic fragment of the ompA protein, which is known to exist in the periplasmic space, increased its susceptibility to [3H]dinitrophenylfluorobenzene only 5.3-times upon disruption of the cell structure. These results indicate that the prolipoprotein accumulated in the presence of globomycin is translocated across the cytoplasmic membrane and interacts with the outer membrane. At the same time, it is attached to the cytoplasmic membrane with its amino-terminal signal peptide in such a way that the amino-terminal portion of the signal peptide containing two lysine residues is left inside the cytoplasm.