Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC50 of approximately 60 microM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 microM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.