Transcription of POX1, the gene encoding peroxisomal acyl-CoA oxidase in the yeast Saccharomyces cerevisiae, is controlled by the carbon source given for cell growth. The gene is repressed in glucose, derepressed in glycerol, and induced in oleate. This regulation is controlled by cis-acting elements in the 5' region of the gene, which bind regulatory proteins. By deletion analysis and DNA band shift assays, we have characterized three such elements in the POX1 upstream sequence, two upstream repression sequences and an upstream activating sequence. Each was able to regulate the transcription of a heterologous gene construct iso-1-cytochrome-c (CYC1)/lacZ.