The DNA sequence of the structural gene (yopE) of one of the Yersinia pseudotuberculosis virulence plasmid-plB1-encoded proteins, Yop5, is presented. The deduced protein showed a molecular weight of 22,971 Daltons. A specific mutant, having a kanamycin-resistance fragment inserted within the yopE gene was no longer virulent for mice. The expression of the Yop5 protein is regulated at the level of transcription by temperature as well as by the Ca2+-concentration of the medium. A significant increase in the level of transcription was not detected until 45 min after a temperature shift from 26 degrees C to 37 degrees C in the absence of calcium; addition of Ca2+ inhibited the expression. The yopE promoter is under positive, as well as negative, plB1-encoded control. The positive function is solely regulated by temperature, while the regulation of the negative function involves at least five different plasmid-encoded gene loci; one of these genes encodes the V-antigen.