The phylogenetic position of ascidians near the base of the chordate tree makes them ideal organisms for evolutionary developmental studies of programmed cell death (PCD). In the present study, the following key features of an apoptotic form of PCD are described in Boltenia villosa: fragmentation of DNA, increases in plasma membrane permeability, decreases in mitochondrial activity, production of reactive oxygen species (ROS), and caspase activation. First, evidence is presented for apoptosis of cells within the ovary. Later in development, during the early phase of larval tail resorption at the beginning of metamorphosis, some notochord nuclei showed DNA fragmentation and their cell corpses were rapidly eliminated from the larval body. In striking contrast to the rapid demise of notochord cells, larval muscle cells persisted for more than a week within developing juveniles. Rhodamine 123 and MTT experiments suggest that mitochondria within some of the resorbed larval tail muscle cells were metabolically active for more than a week. Furthermore, resorbed tail muscle cells contained a muscle-specific intermediate filament, termed p58, despite relatively high levels of ROS activity and the ubiquitination of their plasma membranes at day two. Corpses of larval tail muscle cells containing aggregated pigment granules survived within juveniles for more than a month, in contrast to the rapid elimination of notochord cells. Evidence consistent with the formation of larval muscle cell apoptotic bodies is presented. The most surprising result of the present study was that caspase-8, usually associated with apoptotic signaling, was activated in larval endoderm cells that develop into adult structures. When the present results were compared to features of PCD previously reported in other ascidians, significant species differences in PCD were revealed.