Aims: Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into L(-)-carnitine in Escherichia coli. Methods and Results: The caiC gene was cloned and overexpressed in E. coli and its effect on the production of L(-)-carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI-MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA-transferase activity was also detected in vitro, the main in vivo role of CaiC was found to be the synthesis of betainyl-CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to L(-)-carnitine to be enhanced nearly 20-fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d(+)-carnitine was used as substrate. Conclusions: The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli. Significance and Impact of the Study: A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l-carnitine with Escherichia coli.