Activation of the CYC1 upstream activation site, UAS2, and transcription of several other genes encoding respiratory functions requires the product of the regulatory gene HAP2. We report here the isolation and characterization of a second UAS2 regulatory gene, HAP3. Like mutations in HAP2, a mutation in HAP3 abolishes the activity of UAS2 and prevents growth on nonfermentable carbon sources. The HAP3 gene was cloned and, surprisingly, was found to encode two divergently transcribed, overlapping transcripts: a 570-base RNA and a 3-kilobase (kb) RNA. Chromosomal disruption experiments defined the critical region for HAP3 function to a 1.3-kb segment in which the two transcripts overlap. Analysis of the HAP3 DNA sequence showed that the 570-base transcript could encode a protein of 144 amino acids. Synthesis of the 144-amino-acid protein under regulatory control in vivo demonstrated that this protein is essential for activity of UAS2 as well as for growth on nonfermentable carbon sources. The largest open reading frame in the critical region of the 3-kb transcript is only 86 amino acids. Using site-directed mutagenesis, we demonstrated that the 86-amino-acid open reading frame was not involved in UAS2 activity. The possible role of this 3-kb antisense RNA in HAP3 expression or function is discussed.