The expression of l-asparaginase II (encoded by ansB) in Salmonella enterica was found to be positively regulated by the cAMP receptor protein (CRP) and anaerobiosis. The anaerobic regulation of the S. enterica ansB gene is not mediated by the anaerobic transcriptional activator FNR. This is unlike the situation of the ansB gene of Escherichia coli, which is dependent on both CRP and FNR. To investigate this fundamental difference in the regulation of l-asparaginase II expression in S. enterica, the ansB gene was cloned and the nucleotide sequence of the promoter region determined. Sequence analysis and transcript mapping of the 5′ promoter region revealed a single transcriptional start point (tsp) and two regulatory sites with substantial homology with those found in E. coli. One site, centred –90.5 bp from the tsp, is homologous to a hybrid CRP/FNR (‘CF’) site which is the site of CRP regulation in the E. coli promoter. The other site, centred 40.5 bp upstream of the tsp, is homologous to the FNR binding site of the E. coli promoter. Significantly, however, a single base-pair difference exists in this site, at a position of the related CRP and FNR DNA-binding site consensus sequences known to be involved in CRP versus FNR specificity. Site-directed mutagenesis indicates that this single difference, relative to the homologous E. coli site, results in a CRP binding site and the observed FNR-independent ansB expression in S. enterica. Thus, not only may CRP and FNR sites be interconverted experimentally, with few nucleotide changes, but this has apparently contributed to the evolutionary divergence of ansB regulation in E. coli and S. enterica.