The Drosophila act5C gene has two leader exons at which transcription initiation occurs. In this way two classes of transcripts that are different with respect to the 5'-untranslated sequences are synthesized. Both are present in Drosophila Kc cell mRNA. To define the sequences necessary for transcription from each start point and to determine if each is driven by a separate promoter, 5'-flanking regions from the act5C gene were inserted upstream from the bacterial chloramphenicol acetyltransferase gene and tested for promoter activity by transient assays in Drosophila Kc cells. We show that both leader exons are preceded by separate, functional, promoters. The exon 1 proximal promoter contains at least two regions important for optimal expression. One is at more than 1.9 kb upstream from the exon 1 cap site while the other lies between 1.2 and 0.09 kb of the cap site. The promoter elements necessary for transcription from exon 2 are within 450 bp upstream from its cap site. The data suggest that, in some constructions, transcription initiation at exon 1 inhibits transcription initiation at exon 2. There is a sequence of dyad symmetry which is present upstream from both exon 1 and exon 2 of the form CC(A-rich)6GG. The same sequences are found upstream from many mammalian and chicken actin genes and of the human and mouse c-fos genes, where they are believed to be transcription regulatory sequences.