The emrAB locus of Escherichia coli encodes a multidrug resistance pump that protects the cell from several chemically unrelated antimicrobial agents, e.g., the protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and tetrachlorosalicyl anilide and the antibiotics nalidixic acid and thiolactomycin. The mprA gene is located immediately upstream of this locus and was shown to be a repressor of microcin biosynthesis (I. del Castillo, J. M. Gomez, and F. Moreno, J. Bacteriol. 173:3924-3929, 1991). There is a putative transcriptional terminator sequence between the mprA and emrA genes. To locate the emr promoter, single-copy lacZ operon fusions containing different regions of the emr locus were made. Only fusions containing the mprA promoter region were expressed. mprA is thus the first gene of the operon, and we propose that it be renamed emrR. Overproduction of the EmrR protein (with a multicopy vector containing the cloned emrR gene) suppressed transcription of the emr locus. A mutation in the emrR gene led to overexpression of the EmrAB pump and increased resistance to antimicrobial agents. CCCP, nalidixic acid, and a number of other structurally unrelated chemicals induced expression of the emr genes, and the induction required EmrR. We conclude that emrRAB genes constitute an operon and that EmrR serves as a negative regulator of this operon. Some of the chemicals that induce the pump serve as its substrates, suggesting that their extrusion is the natural function of the pump.