The solitary ascidian Halocynthia roretzi possesses three types of muscle: the larval tail striated muscle, the adult heart striated muscle, and the adult body wall smooth muscle. The troponin complex is observed in all types of muscle, and the isoform sequences and expression patterns of two of the three troponin components, troponins C and T, have been reported. In this study, we have determined cDNA sequences of the three TnI isoforms from H. roretzi. One of the three isoforms (adult TnI), expressed in adult body wall smooth muscle and heart muscle, was composed of 173 amino acids, being similar to vertebrate fast and slow skeletal TnIs in length. The other two isoforms (larval TnI alpha and TnIbeta) were isolated from a cDNA library of larvae. Both larval TnIs were composed of 142 amino acids, with truncation amounting to ca. 30 amino acid residues at the C-termini. These larval TnIs are the smallest known TnIs. The position of the last intron of these TnIs was also determined. When compared with vertebrate TnI genes, the last intron of the ascidian adult TnI gene is located at 6 nucleotides downstream, and the introns of the two larval TnIs are positioned at 9 nucleotides upstream. These results suggest that H. roretzi TnI is encoded by at least three genes.