Newborn Sprague-Dawley rats received a single dose of 2 Gy X-rays and were killed 6 hr later. Dying cells were characterized by extreme chromatin condensation and nuclear fragmentation. Dying cells were distributed in the primary and secondary germinal zones and in other brain regions. Among these latter, dying cells occurred in the cortical layers of the olfactory bulb, layers II-III and VIb of the neocortex, piriform and entorhinal cortex, stratum oriens and pyramidale of the hippocampus, striatum, thalamus, amygdala, brainstem, internal granular layer of the cerebellum, and cerebral and cerebellar white matter. Dying cells were immature cells, neurons and glial cells (including radial glia). In-situ labeling of nuclear DNA fragmentation identified individual cells bearing fragmented DNA. Since the number of cells stained with this method was larger than the number of dying cells, as revealed with current histological techniques, it is suggested that nuclear DNA fragmentation precedes chromatin condensation and nuclear fragmentation in X-ray-induced apoptosis. Furthermore, agarose gel electrophoresis of extracted DNA from irradiated brains showed a "ladder" pattern which is typical of internucleosomal DNA fragmentation and endonuclease activation.