Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine-sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS-PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai-fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half-sites separated by 13 bp, and centred at -70 and -127 from the fix transcription start site. They were found to overlap the two low-affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai-fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP-CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.