To understand the molecular basis for the regulation of rat A2a adenosine receptor (A2a-R) expression, we have characterized the rat A2a-R gene and defined its promoter regions. Through a combination of restriction mapping and sequence analysis, we have demonstrated that the rat A2a-R gene is composed of two exons interrupted by a 7.2-kb intron. Primer extension and RNase protection on RNA isolated from PC12 cells suggested that the A2a-R gene encoded two clusters of alternative transcripts. The most upstream transcription start site was designated as +1. The sequence of the proximal 1.5 kb of 5'-flanking region demonstrated no potential TATA box, CCAAT box, or initiator element in the appropriate location. Varying lengths of 5'-flanking regions were inserted into a transient expression vector (pGL2-basic), which contained bacterial luciferase as the reporter gene, to determine its promoter region(s) in PC12 cells, CHOP cells, and C6 cells. Consistent with two clusters of transcription start sites, two independent functional promoter regions (designated P1, -67/-1; and P2, +272/+304) for the rat A2a-R gene were identified. Although both promoters are in use in PC12 cells, only P2 is active in CHOP cells, indicating possible cell line-specific usage of these two promoters.