In Saccharomyces cerevisiae glycerol utilization is mediated by two enzymes, glycerol kinase (Gut1p) and mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). The carbon source regulation of GUT1 was studied using promoter-reporter gene fusions. The promoter activity was lowest during growth on glucose and highest on the non-fermentable carbon sources, glycerol, ethanol, lactate, acetate and oleic acid. Mutational analysis of the GUT1 promoter region showed that two upstream activation sequences, UAS(INO) and UAS(ADR1), are responsible for approximately 90% of the expression during growth on glycerol. UAS(ADR1) is a presumed binding site for the zinc finger transcription factor Adr1p and UAS(INO) is a presumed binding site for the basic helix-loop-helix transcription factors Ino2p and Ino4p. In vitro experiments showed Adr1 and Ino2/Ino4 protein-dependent binding to UAS(ADR1) and UAS(INO). The negative regulator Opi1p mediates repression of the GUT1 promoter, whereas the effects of the glucose repressors Mig1p and Mig2p are minor. Together, the experiments show that GUT1 is carbon source regulated by different activation and repression systems.