The lcrGVH operon of plasmid pCD1 in Yersinia pestis KIM encodes the virulence-associated V antigen, the regulatory protein LcrH, and LcrG, a protein of undefined function. In this study we sequenced lcrGVH and analyzed it for transcription initiation sites. There were three open reading frames within the sequence, 288, 981, and 507 bases in length, which could encode proteins with molecular weights and isoelectric points corresponding to those of LcrG, LcrV (V antigen), and LcrH, respectively. The predicted LcrV protein lacked an N-terminal signal sequence; however, an internal signallike sequence was present. An Escherichia coli-like promoter consensus sequence was detected upstream from lcrG. Primer extension analysis showed that (i) the transcriptional start site for lcrGVH was spaced only three bases upstream from the nearest ATG potential start site, raising the possibility that Y. pestis may use an alternate initiation codon for the V operon; (ii) there was much more primer-extended product in yersiniae grown in the absence of Ca2+ than in its presence, showing for the first time that lcrGVH is regulated at the transcriptional level by Ca2+; (iii) no separate lcrV initiation was detected, indicating that the V antigen is expressed from messages initiating at lcrG; and (iv) a non-Ca2+-regulated transcriptional start site was found upstream from lcrH, suggesting that the LcrH protein is expressed constitutively. However, two-dimensional gel analysis showed that net LcrH expression was regulated by Ca2+. We propose that lcrH lies within two differentially regulated operons, its own and lcrGVH.