In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)–CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.