Transcription of the ansB gene, encoding L-asparaginase II, is positively regulated by cAMP receptor protein (CRP) and by the product of the fnr gene, the FNR protein. These global regulatory proteins mediate the expression of ansB in Escherichia coli in response to carbon source and to anaerobiosis, respectively, and are required concurrently for optimal ansB expression. The mechanism whereby CRP and FNR interact co-operatively with the ansB promoter to achieve transcription has not previously been established. We have utilized an ansB'-'lacZ fusion, in conjunction with deletion analysis and site-directed mutagenesis, to identify two sites which interact with these regulatory proteins in the ansB promoter. The first is an FNR site, centred 41.5 bp upstream of the major transcriptional start site. The second site, located 28 bp upstream of the FNR site, is the site of CRP regulation. This site is homologous to both the CRP and FNR binding-site consensus sequences and may respond to both CRP and FNR. The concurrent requirement for CRP and FNR for optimal expression of ansB may be explained if, first, essentially no transcription occurs unless the FNR is bound at the downstream site, and, second, the level of transcription when FNR alone is present is enhanced when CRP binds at the upstream site.