We have carried out a functional analysis of invl and invJ, two Salmonella typhimurium genes required for this organism to gain access to cultured mammalian cells. These genes are located immediately down-stream of invC, a previously identified gene also required for bacterial invasion. Non-polar mutations in either of these genes rendered S. typhimurium severely defective for entry into cultured epithelial cells, although these mutations did not affect the ability of these organisms to attach to those cells. Nucleotide sequence analysis revealed that the invl and invJ genes encode proteins with molecular weights of 18,077 and 36,415, respectively. Polypeptides of similar sizes were observed when these genes were expressed in a bacteriophage T7 RNA polymerase-based expression system. Comparison of the predicted sequences of invl and invJ with translated sequences in the existing databases indicated that these proteins are identical to the previously identified S. typhimurium SpaM and SpaN proteins. Further analysis of these sequences revealed regions of homology between Invl and the N-terminus of IpaB of Shigella spp. and between InvJ and EaeB of enteropathogenic Escherichia coli. Localization studies by immunoblot analysis indicated that InvJ is secreted to the culture supernatant, a surprising finding since this protein also lacks a typical signal sequence. Mutations in invG and invC, two members of the Salmonella inv locus, effectively prevented the transport of InvJ to the culture supernatant. Thus, InvJ is the first identified target of the protein secretion apparatus encoded in the inv locus and therefore a candidate to have effector functions related to bacterial entry.