Fatty acid desaturation catalyzed by fatty acid desaturases requires molecular oxygen (O(2)). Saccharomyces cerevisiae cells derepress expression of OLE1 encoding Delta9 fatty acid desaturase under hypoxic conditions to allow more-efficient use of limited O(2). It has been proposed that aerobic conditions lead to repression of OLE1 by well-established O(2)-responsive repressor Rox1p, since putative binding sequences for Rox1p are present in the promoter of OLE1. However, we revealed in this study that disruption of ROX1 unexpectedly did not affect the O(2) repression of OLE1, indicating that a Rox1p-independent novel mechanism operates for this repression. We identified by promoter deletion analysis the 50-bp O(2)-regulated (O2R) element in the OLE1 promoter approximately 360 bp upstream of the start codon. Site-directed mutagenesis of the O2R element showed that the putative binding motif (5'-GATAA-3') for the GATA family of transcriptional factors is important for O(2) repression. Anaerobic derepression of OLE1 transcription was repressed by unsaturated fatty acids (UFAs), and interestingly the O2R element was responsible for this UFA repression despite not being included within the fatty acid-regulated (FAR) element previously reported. The fact that such a short 50-bp O2R element responds to both O(2) and UFA signals implies that O(2) and UFA signals merge in the ultimate step of the pathways. We discuss the differential roles of FAR and O2R elements in the transcriptional regulation of OLE1.