A novel peptidoglycan-linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species-related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild-type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild-type strain. comL is a single-copy gene and maps downstream of the previously described comA gene It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5′ end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA′ ). In E. coli, cloned ComL can be labelled with [3H]-palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseriameningitidis as well as in several non-pathogenic Neisseria species.