SummaryWe have carried out a functional analysis ofinvlandinvJ, twoSaimonella typhimuriumgenes required for this organism to gain access So cultured mammalian cells. These genes are located immediately downstream ofinvC, a previously identified gene also required for bacterial invasion. Non‐polar mutations in either of these genes renderedS. typhimuriumseverely 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 theinvlandinvJgenes encode proteins with molecular weights of 18077 and 36415, 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 identifiedS. typhimuriumSpaM and SpaN proteins. Further analysis of these sequences revealed regions of homology between Invl and theN‐terminus of lpaB ofShigellaspp. and between InvJ and EaeB of enteropathogenicEscherichia 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 ininvGandinyC, two members of theSalmonella invlocus, 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