Growth conditions influenced DNA methylation in cultured cerebellar granule cells, as indicated by immunocytochemical analysis with monoclonal antibodies raised against 5-methylcytidine. In cultures grown under suboptimal conditions, i.e. in medium containing 10 instead of 25 mM K+, a substantial reduction in both the number of immunopositive cells and the intensity of immunostaining occurred at 4 days in vitro (DIV), a time which preceded the appearance of the morphological features of apoptosis. These results suggest that a reduction in DNA methylation is one of the biochemical events associated with the 'condemned phase' of apoptosis, in which granule cells grown under suboptimal conditions become committed to death.