Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position -41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism.