This paper describes the mutagenesis of the pYV plasmid from Yersinia enterocolitica W22703 (serotype O:9) with Tn2507, a new element generating operon fusions. Analysis of the mutants allowed the identification of an additional Yop protein called Yop20 and the mapping of yop20, yop44, yop48, and lcrV, the gene encoding the V antigen. The last gene appeared to be part of an operon that also may contain yop37 and yop44. At 37 degrees C, mutants affected in this operon grew poorly, irrespective of the presence of Ca2+, or they even died in the presence of Ca2+. This operon is thus involved in the regulation by Ca2+, and we called it car, for Ca2+ regulation. It is presumably the Y. enterocolitica counterpart of the lcrGVH operon of Yersinia pestis. Transcription of yop20 and of the car operon was strongly regulated by temperature and only slightly by calcium. Hence, these genes behaved like the other genes of the yop regulon. Mutants affected in yop20 or in yop48 were markedly less virulent for the desferrioxamine-treated mouse than was the parental strain. Yop20 and Yop48 thus probably are Yersinia virulence factors.