Previous studies have demonstrated a correlation between the ability of isolates of Yersinia enterocolitica to cause disease and to invade tissue culture cells in vitro. Two genes, inv and ail, isolated from a pathogenic strain of Y. enterocolitica have each been shown to confer this invasive phenotype upon Escherichia coli. Eighty pathogenic, invasive isolates studied by Miller et al. (Infect. Immun. 57:121-131, 1989) contained sequences homologous to both of these genes. Thirty-five nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. We investigated inv-homologous sequences from four nonpathogenic isolates. Recombinant clones of these inv-homologous sequences did not confer the invasive phenotype upon E. coli. No RNA transcripts capable of encoding a full-length Inv protein were detected in the four noninvasive Yersinia strains. When the inv gene from a pathogenic isolate was introduced into two of these strains, the resulting transformants invaded tissue culture cells in vitro. The inv gene was transcribed in a pathogenic Yersinia isolate grown at 30 degrees C but not at all in these cells grown at 37 degrees C. The production of RNA transcripts homologous to inv in transformants was not regulated by temperature to the same degree as was seen for pathogenic isolates. We conclude that the inv gene in nonpathogenic strains of Y. enterocolitica is nonfunctional. Y. enterocolitica isolates epidemiologically linked to disease contain both a functional inv gene and a functional ail gene. Environmental isolates not associated with disease have a nonfunctional inv gene and no ail gene.