The mod (chlD) locus at 17 min on the Escherichia coli chromosome encodes a high-affinity molybdate uptake system. To further investigate the structure and regulation of these genes, the DNA region upstream of the previously identified modBC (chlJD) genes was cloned and sequenced. A single open reading frame, designated modA, was identified and appears to encode a periplasmic binding protein for the molybdate uptake system. To determine how the mod genes are regulated in response to molybdate, nitrate, and oxygen, we constructed a series of mod-lacZ operon fusions to the upstream region and introduced them in single copy onto the E. coli chromosome. Whereas molybdate limitation resulted in elevated mod-lacZ expression, neither oxygen nor nitrate had any significant effect on gene expression. A regulatory motif, CATAA, located at the modA promoter was identified and shown to be required for molybdate-dependent control of the modABCD operon. Mutations within this sequence resulted in nearly complete derepression of gene expression and suggest that transcription of the operon is mediated by a molybdenum-responsive regulatory protein.