The FadR protein of Escherichia coli has been shown to play a dual role in transcription of the genes of bacterial fatty acid metabolism. The protein acts as a repressor of beta-oxidation and an activator of unsaturated fatty acid synthesis. FadR DNA binding is antagonized by long chain acyl-CoAs, and thus FadR acts as a sensor of fatty acid availability in the environment. When viewed from a genomic viewpoint, FadR proteins are unusual in that the DNA binding domain is very highly conserved among FadR-containing bacteria, whereas the C-terminal acyl-CoA binding domain shows only weak conservation. To further our understanding of the role of FadR in bacterial lipid metabolism we have examined the in vivo and in vitro properties of a diverse set of FadR proteins expressed in E. coli. In addition to E. coli FadR the proteins examined were those of Salmonella enterica, Vibrio cholerae, Pasteurella multocida, and Haemophilus influenzae. These FadR proteins were found to differ markedly in their effects on repression and induction of beta-oxidation in E. coli and in their acyl-CoA binding abilities as measured by isothermal titration calorimetry. The E. coli and S. enterica proteins were the most similar, although they differed in their effects on utilization of oleic acid and acyl-CoA binding affinities, whereas the P. multocida and H. influenzae proteins showed only weak repression and poor acyl-CoA binding affinities. The V. cholerae FadR was strikingly superior to the other proteins in the amplitude of its regulatory response, and it bound long chain acyl-CoAs appreciably more strongly than the E. coli and S. enterica proteins. The significance of these findings is discussed in view of the protein sequences and the physiological niches occupied by these organisms.