The evolutionary history of the diverse lifestyles adopted by urochordates has attracted intense interest because it may effect the evolutionary history of vertebrates. Here, we report the complete mitochondrial (mt) DNA sequence of the pelagic thaliacean doliolid Doliolum nationalis. The doliolid mt genome shares the unusual tRNAs of trnM(uau) and trnG(ucu) with other ascidians, such as Halocynthia and Ciona. On the other hand, the gene order of the doliolid mt genome is significantly different from that of any ascidian species or vertebrate reported to date. Phylogenetic analyses of the amino acid sequences of 12 protein-coding genes strongly support the sister-grouping of doliolids and the Phlebobranch ascidian Ciona, with the Stolidobranch ascidian alocynthia as the outgroup, thereby providing strong support for the paraphyly of ascidians, as has been suggested by 18S rDNA studies. Given the paraphyletic nature of ascidians, it seems likely that the common ancestor of ascidians and thaliaceans was sessile, as are the present-day ascidians, and that the thaliaceans subsequently evolved a pelagic lifestyle.