Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons. IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines. We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP. The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor. Whether CarP directly binds to this site or exerts its protective effect indirectly is not yet known. In the absence of IHF (himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream. Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity. The absence of both these effects in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.