The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone. The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal. To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system. The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system. An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation. L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts. Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P. In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP). A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release. The outer membrane-specific sorting signal was essential for the in vitro release of L10P. Furthermore, L10P released in vitro was specifically incorporated into the outer membrane. These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy.