Examination of 69 strains of Yersinia enterocolitica which represented 20 serotypes and nontypable isolates for HeLa cell infectivity by a roller tube technique that provided a quantitative index of infection showed that infectivity (index, greater than 3.50) was confined to strains of serotypes O:8, O:3, O:5, 27, O:9, O:1, O:1,2,3, O:2,3, O:4,32, and O:21. All strains that were HeLa positive were sucrose positive and negative for salicin, esculin, rhamnose, raffinose, melibiose, alpha-methylglucoside, and citrate. All HeLa-negative strains were either sucrose and salicin positive or were sucrose negative. Twenty-one strains were examined for virulence by the ability to produce guinea pig conjunctivitis and diarrhea in mice. Positive strains were limited to those that were HeLa positive and were autoagglutination positive and calcium dependent at 35 degrees C. There was no association between virulence and the ability to produce enterotoxin measured by the infant mouse assay. Loss of autoagglutinability and calcium dependency was accompanied by loss of virulence, but HeLa cell infectivity was unchanged. The results suggest that at least two properties are necessary for virulence: the presence of the V and W antigens, mediated by the same plasmid as autoagglutination and calcium dependency, and an invasive factor demonstrated in vitro by HeLa cell infectivity. These virulence properties are found in only certain biotypes of Y. enterocolitica.