Sequences required for Saccharomyces cerevisiae actin gene transcription were mapped and compared to the regulatory region of the actin gene from a thermophilic fungus, Thermomyces lanuginosus. Two CCAAT motifs conserved in position in these two species could be mutated without affecting promoter activity, regardless of whether the yeast were grown in fermentable or non-fermentable carbon sources. Two TATA-like sequences and an upstream activation sequence (UAS) composed of multiple elements were identified. The contribution of sequence motifs within these elements to UAS activity varied depending on the carbon source. The Thermomyces gene contains sequences highly homologous to this UAS, but in the opposite orientation.