A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for half-maximal binding (K0.5) was calculated to be 1 microM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (K0.5, 10 nM) and much more effective than biotin in the DNA-binding assay (K0.5 1.1 nM versus 1 microM). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.