Experiments were performed to study whether the synthesis of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli is affected by glucose. The CFA/1-producing strain H-10407 (078:H11:CFA/I) was grown in CFA medium containing various concentrations of glucose. Addition of 1% glucose into the medium resulted in a pronounced decrease in CFA/I production by H-10407 as assessed by ELISA, hemagglutination, and electron microscopy. The repressive effect of glucose was reversed by the addition of 10 mM cAMP to the medium. Examination of the promoter sequence of the cfaA gene of the CFA/I operon revealed a consensus binding site for the catabolite activator protein-cAMP complex. With a reporter plasmid containing a fusion of the cfaA promoter, a portion of the cfaA gene, and the lacZ gene, it was shown that the activity of this promoter was influenced by glucose. In a wild-type E. coli strain, addition of 0.5% glucose to the growth medium diminished the promoter activity more than 70%. The cfaA promoter also exhibited a lower level of activity in cya (adenyl cyclase) and crp (cAMP receptor protein) mutants than in the wild-type strain. The addition of 10 mM cAMP resulted in a marked increase in the expression from the cfaA promoter in the cya but not in the crp mutant. These results suggest that the suppressive effect of glucose in the CFA/I system is mediated via the mechanism of catabolite repression through the cfaA promoter of the CFA/I operon.