Fatty acyl-CoA thioesters are essential intermediates in lipid metabolism. For many years there have been numerous conflicting reports concerning the possibility that these compounds also serve regulatory functions. In this review, we examine the evidence that long-chain acyl-CoA is a regulatory signal that modulates gene expression. In the bacteria Escherichia coli, long-chain fatty acyl-CoA bind directly to the transcription factor FadR. Acyl-CoA binding renders the protein incapable of binding DNA, thus preventing transcription activation and repression of many genes and operons. In the yeast Saccharomyces cerevisiae, genes encoding peroxisomal proteins are activated in response to exogenously supplied fatty acids. In contrast, growth of yeast cells in media containing exogenous fatty acids results in repression of a number of genes, including that encoding the delta9-fatty acid desaturase (OLE1). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase increases the activation of transcription of a fatty acid-responsive promoter, whereas coexpression with thioesterase decreases the fatty acid-mediated response. Conflicting data exist in support of the notion that fatty acyl-CoA are natural ligands for peroxisomal proliferator-activated receptor alpha (PPARalpha).