A virulent plasmid-containing strain and an isogenic plasmid-free avirulent derivative strain of Y. pseudotuberculosis were compared with respect to the interaction with cultured HeLa cells. No difference in the ability to adhere to HeLa cells was observed. Both strains invaded the HeLa cells to the same degree. The internalized bacteria did not multiply over a 24-h period. When incubated at 37 degrees C HeLa cells infected with the virulent plasmid-containing strain YPIII(pIB1) showed morphological changes in contrast to the avirulent plasmid-free strain YPIII which showed no such effect. This cytotoxic effect was recorded after about 2 hours of incubation at 37 degrees C. This event could be prevented by addition of gentamicin 1 hour before the predicted time of the morphological changes of the HeLa cells. A number of transposon Tn5 derived plasmid mutants were analyzed and a correlation was found between virulence and ability to induce cytotoxicity. The cytotoxic effect was not absolutely linked to Ca2+-dependence because one Ca2+-independent mutant was found to be cytotoxic. This mutant was able to express the temperature-inducible plasmid-coded outer membrane proteins (YOPs) as well as the V-antigen. On the molecular level a strong correlation was found between expression of some of the YOPs, the V-antigen and the cytotoxic effect. However, one of these YOPs, YOP1, could be eliminated as being an important determinant of cytotoxicity because an insertion mutant of the corresponding structural gene was still able to induce cytotoxicity.