Apoptosis is one form of physiological or programmed cell death responsible for the selective elimination of various cell types during development. We have observed and characterized a delayed-type of neurotoxicity induced in cultured cerebellar granule neurons by diphenylhydantoin. Diphenylhydantoin toxicity of cerebellar granule neurons is time and concentration dependent. Morphological studies using Nomarski optics and staining with the fluorescent dye Hoechst 33258 demonstrate that diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is associated with cytoplasmic blebbing, heterochromatic clumping and condensation of chromatin that precede cell death. Unlike glutamate toxicity (excitotoxicity) diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is attenuated by actinomycin D and cycloheximide, and is associated with nucleosomal size DNA fragmentation. Since we have previously reported that depolarization of cultured cerebellar granule neurons with high concentrations of K+ promotes the survival of these neurons by blocking apoptosis, we examined the effects of diphenylhydantoin on the K(+)-evoked increase in intracellular calcium. Using microfluorimetry and fura-2 to measure intracellular calcium we found that neurotoxic concentrations of diphenylhydantoin markedly reduce the increase in intracellular calcium associated with elevated extracellular potassium. Taken together, these data demonstrate that exposure of cultured cerebellar granule neurons to pharmacologically relevant concentrations of diphenylhydantoin results in a delayed type of neurotoxicity characterized by the biochemical and morphological features of apoptosis.