We have isolated and determined the complete nucleotide sequence of the gene that encodes the 248 amino acid residue fibroblast tropomyosin, TM-4. The TM-4 sequence is encoded by eight exons, which span approximately 16,000 bases. The position of the intron-exon splice junctions relative to the final transcript are identical to those present in other vertebrate tropomyosin genes and the Drosophila melanogaster TMII gene. We have found no evidence that the rat TM-4 gene is alternatively spliced, unlike all the other tropomyosin genes from multicellular organisms that have been described. Typical vertebrate tropomyosin genes contain some, or all, of alternatively spliced exons 1a and 1b, 2a and 2b, 6a and 6b, and 9a, 9b, 9c and 9d in addition to common exons 3, 4, 5, 7 and 8. The rat fibroblast TM-4 mRNA is encoded by sequences most similar to exons 1b, 3, 4, 5, 6b, 7, 8 and 9d. Two exon-like sequences that are highly similar to alternatively spliced exons 2b and 9a of the rat beta-tropomyosin gene and the human TMnm gene have been located in the appropriate region of the gene encoding rat fibroblast TM-4. However, several mutations in these sequences render them non-functional as tropomyosin coding exons. We have termed these exon-like sequences, vestigial exons. The evolutionary relationship of the rat TM-4 gene relative to other vertebrate tropomyosin genes is discussed.