We isolated and delimitated the Drosophila ras2 promoter region, determined its sequence and mapped the transcription units expressed in this region. The results showed that the Drosophila ras2 gene is flanked by another transcription unit, which codes for two larger transcripts, 2.5 and 2.9 kb long. Orientation experiments, in which sense and antisense RNA probes were used, revealed that both these and the ras2 transcripts are synthesized from different DNA strands. Thus, the flanking transcription unit is in the opposite polarity relative to the ras2 gene. The transcription start sites of the ras2 gene and the flanking transcription unit were determined by external primer extension with T4 DNA polymerase and by RNAase-protection assay and were found to be only 94 nucleotides apart. Apparently, the Drosophila ras2 promoter is a bidirectional promoter. Nucleotide sequence analysis revealed that the 5'-end of the ras2 transcript is within an inverted repeat of the insect cap box. TATA- and GC-like boxes were also found. Analysis of direct and inverted repeats in the promoter region suggested that it is asymmetrical. To demonstrate promoter activity, each side of the ras2 bidirectional promoter was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and tested by transfecting Drosophila Schneider 2 culture cells. Significant CAT activity was obtained with both transcription fusions.