The Saccharomyces cerevisiae COX5b gene is regulated at the level of transcription by both the carbon source and oxygen. To define the cis-acting elements that underlie this transcriptional control, deletion analysis of the upstream regulatory region of COX5b was performed. The results of the study suggest that at least four distinct regulatory sites are functional upstream of the COX5b transcriptional starts. One, which was precisely defined to a region of 20 base pairs, contains two TATA-like elements. Two upstream activating sequences (UAS15b and UAS25b) and an upstream repression sequence (URS5b) were also found. Each of the latter three elements was able either to activate (UAS15b and UAS25b) or to repress (URS5b) the transcription of a heterologous yeast gene. Further analysis revealed that UAS15b is the site of carbon source control and may be composed of two distinct domains that act synergistically. URS5b mediates the aerobic repression of COX5b and contains two sequences that are highly conserved in other yeast genes negatively regulated by oxygen.