The muscle tropomyosin I gene of Drosophila melanogaster undergoes alternative splicing in different muscles of the fly to generate two isoforms of the same protein. We report here the structural analysis and DNA sequence of the tropomyosin I gene. The gene spans 5 kilobase of DNA and is comprised of five exons and four introns. Exon 4 is alternatively spliced in RNA of different muscle, resulting in two isoforms of the same protein. The gene lacks a "TATA" box homology at the map position; it is usually found in the vast majority of eukaryotic genes characterized thus far. Instead, a series of three alternating TG stretches are located upstream from the site of initiation of transcription. The gene encodes a 5' untranslated leader of 103 base pairs, and the 3' untranslated region comprises between 30 and 50% of the transcripts. The DNA sequence is extremely G + C rich in the protein coding regions of the gene, and A + T rich in the non-coding, flanking, and intron regions. The DNA sequence upstream of the acceptor sites in the two introns which are subject to alternative splicing displays a stretch of homology which is noted. The 3' untranslated region of the fifth exon contains multiple polyadenylation sites. The 284 amino acid protein encoded by the gene is split by introns between residues 198/199 and 257/258. These sites correlate closely with two important functional domains in the tropomyosin molecule. A comparison of the first 257 amino acids and the carboxyl-terminal 27 amino acids of the Drosophila and vertebrate tropomyosins together, shows two distinct and mutually exclusive classes for these domains. The functional significance of the Drosophila tropomyosin isoforms is discussed.