Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of beta-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of beta-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3'-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.