The plasmid pCD1 is required for expression of the low-calcium response (LCR), virulence, and production of V antigen in Yersinia pestis KIM. Five independent mutants constitutive for the LCR at 37°C (Lcr(c)) were obtained through ethyl methanesulfonate mutagenesis followed by ampicillin enrichment. A sixth, spontaneous mutant was obtained directly through ampicillin enrichment. These mutants failed to grow at 37°C regardless of calcium concentration and produced V antigen constitutively at this temperature. All six mutations were located on pCD1. One mutation was mapped to a 1-kilobase region of lcrA. Based on complementation mapping of this mutation, the lcrA locus was divided into two new loci, lcrD and lcrE. This mutation, lcrE1, did not alter the transcription of other genes in the LCR region and was cis-recessive to lcr mutations. Several lower-molecular-weight outer membrane proteins which were observed in the parent strain grown at 37°C in the presence of 2.5 mM calcium were reduced in quantity or absent from the mutant strain.