An evolutionary change in development was studied in two closely related ascidian species, one exhibiting a conventional tadpole larva and the other a modified tailless larva. Interspecific hybridization restores chordate features to the tailless larva. The zinc finger gene Manx is expressed in cells that generate chordate features in the tailed species but is down-regulated in the tailless species. Manx expression is restored in hybrid embryos. Antisense oligodeoxynucleotide treatment inhibited Manx expression and chordate features in hybrid embryos, which suggests that Manx is required for development of the chordate larval phenotype in ascidians.