The basic Yop2b protein, encoded by the virulence plasmid pIBI of Yersinia pseudotuberculosis, is produced under Ca2+-deficient conditions. A mutant deleted for the entire yopH gene, which encodes the Yop2b protein, was found to be avirulent. Virulence could be restored by trans-complementation. The DNA-sequence of yopH predicted a 50 737 D polypeptide lacking a typical signal peptide. Transcription of yopH is regulated by both temperature and Ca2+-concentration. Mutations within the region of the virulence plasmid known to be involved in regulating gene expression in response to Ca2+ abolished transcription of yopH. Other temperature-sensitive mutations in the Ca2+-regulatory locus showed a high level of transcription regardless of Ca2+-concentration. These responses were similar to those of the yopE gene. The promoter region of the yopE and yopH genes were compared and four conserved motifs identified.