Neocarzinostatin (NCS) is a naturally occurring enediyne antitumor agent that produces single- and double-strand breaks in cellular DNA. We have previously shown that treatment of human (SK-N-SH) and murine (NB41A3) neuroblastoma cells with NCS results in cell death for a subpopulation within the culture. The remaining cells undergo mitotic arrest with morphological differentiation along glial lines. Further investigation of cell death induced by this agent demonstrates that within 24 hr after a single one hr exposure to submicromolar concentrations of NCS, susceptible cells of both lines decrease in size, round up, detach from the culture surface and fragment in the overlying medium. This cytotoxicity is attenuated by the addition of cycloheximide (in NB41A3 cells) or aurintricarboxylic acid (in NB41A3 and SK-N-SH cells). Fluorescence and electron microscopic examination of the nonadherent cells reveals the chromatin condensation and fragmentation characteristic of apoptosis. Examination of the time course of DNA cleavage reveals that despite the presence of alkaline elution-detectable DNA cleavage, oligonucleosomal-sized DNA fragments are not demonstrable by gel electrophoresis immediately after a 1-hr incubation with the drug (1.6-10,000 nM). However, by 6 hr after treatment, DNA ladders are in evidence at all concentrations of NCS. These results suggest that the oligonucleosomal cleavage of DNA seen after NCS treatment is associated with apoptosis, rather than being the direct result of the strand-cleaving effects of the drug itself.