Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca(2+)/Mg(2+)-dependent nuclease and a 40-kD Zn(2+)-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn(2+), and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.