The catabolic pathway of N-acetylglucosamine (GlcNAc) in Candida albicans is an important facet of its pathogenicity. One of the pathway genes, encoding glucosamine-6-phosphate deaminase (NAG1) is transcriptionally regulated by GlcNAc. Sequence analysis of a 4-kb genomic clone containing NAG1 indicates that this gene is part of a cluster containing two other genes of the GlcNAc catabolic pathway, i.e., DAC1, GlcNAc-6-phosphate deacetylase, and HXK1, hexokinase. All three genes are temporally and coordinately induced by GlcNAc suggesting a common regulatory mechanism for these genes. The NAG1 promoter is up-regulated when induced by GlcNAc in C. albicans but not in Saccharomyces cerevisiae. In vivo analysis of the deletion constructs delineated the minimal promoter to -130 bp and mapped two regions at -200 and -400 bp upstream of +1 (ATG) responsible for GlcNAc induction. Gel mobility-shift assays and "footprinting" (DNase protection method) analyses revealed two regions, 5'-GGAGCAAAAAAATGT 3' (-164 to -150, box A) and 5'-ACGGTGAGTTG 3' (-291 to -281, box B), that are recognized and bound by at least two inducible activator proteins directing the regulation of gene expression.