The Escherichia coli cytR-encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism. The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene. A chromosomal cytR-lacZ+ operon fusion was isolated and used to study the regulation of cytR. We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex. Footprinting studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter. A previously described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed. DNA sequence analysis of the cytR* mutation revealed a G-C to an A-T base pair transition at position -34 bp relative to the translational initiation site of cytR. This point mutation activates a cryptic promoter that is stronger than the wild-type cytR promoter and leads to overproduction of the CytR repressor.