The central nervous system (CNS) of the ascidian tadpole larva consists of only 370 cells, yet it develops similarly to the elaborate vertebrate CNS. There are two phases in ascidian CNS development: an early phase, in which cells of mixed neuronal and non-neuronal (ependymal) fates are formed, and a late phase, in which only additional ependymal cells are produced. Proliferating cell nuclear antigen (PCNA), the auxiliary factor for DNA polymerase , is expressed zygotically in the developing CNS. Here we employ antisense oligonucleotides directed against PCNA mRNA to determine the role of late dividing ependymal cells in embryos of the ascidian Styela clava. An antisense oligonucleotide beginning at the translation start site in PCNA mRNA was effective in inhibiting zygotic PCNA accumulation. In normal tadpoles, the head is located in the same anteroposterior plane as the tail, which promotes larval swimming and dispersal. Embryos treated with PCNA antisense oligonucleotides developed into boomerang-shaped tadpoles with their heads positioned at an acute angle with respect to their tails, causing them to swim in circles rather than in the usual forward direction. Further studies showed that PCNA inhibition arrested DNA synthesis and induced nuclear DNA fragmentation typical of programmed cell death in the developing CNS. No other defects were apparent in these abnormal tadpoles, and they were eventually able to metamorphose into juveniles. The results suggest that PCNA and late dividing ependymal cells are required for normal CNS development and larval morphogenesis in ascidians.