The Yersinia pestis V antigen is necessary for full induction of low-calcium response (LCR) stimulon virulence gene transcription, and it also is a secreted protein believed to have a direct antihost function. We made four nonpolar deletions in lcrV of Y. pestis to determine if secretion, regulation, and virulence functions could be localized within the V antigen (LcrV). Deletion of amino acids 25 to 40 caused secretion of LcrV to be decreased in efficiency; however, removal of residues 108 to 125 essentially abolished LcrV secretion. Neither mutation had a significant effect on LCR regulation. This showed that LcrV does not have to be secreted to have its regulatory effect and that the internal structure of V antigen is necessary for its secretion. Both mutants were avirulent in mice, showing that the regulatory effect of LcrV could be separated genetically from its virulence role and raising the possibility that residues 25 to 40 are essential for the virulence function. This study provides the best genetic evidence available that LcrV per se is necessary for the virulence of Y. pestis. The repressed LCR phenotype of a mutant lacking amino acids 188 to 207 of LcrV raised the possibility that the deleted region is necessary for regulation of LCR induction; however, this mutant LcrV was weakly expressed and may not have been present in sufficient amounts to have its regulatory effect. In double mutants containing this mutant lcrV and also lacking expression of known LCR negative regulators (LcrG, LcrE, and LcrH), full induction of the LCR occurred in the absence of functional LcrV, indicating that LcrV promotes induction not as an activator per se but rather by inhibiting negative regulators.