Hyperthermia is an efficient means of inducing cell death in vivo and in vitro. Among human neoplastic cells, HeLa S3 cells are susceptible to heat injury when exposed to long duration moderate hyperthermia (41.5 C), conditions that are reproducible and sustainable in the clinical setting. Hence, HeLa S3 cells are a useful substrate for evaluation of hyperthermic injury in human neoplasia. Previous studies have demonstrated a consistent response of HeLa S3 cells to moderate hyperthermia: spontaneous premature condensation of chromosomes during heat exposure in S phase followed by apparent nuclear fragmentation and, inevitably, cell death. To further characterize the morphological features of this process, HeLa S3 cells grown in suspension at 37 C were heated for 4, 8, 12, or 16 hours at 41.5 C and harvested in glutaraldehyde for electron microscopic evaluation. Compared with untreated controls, heated samples exhibited a characteristic pattern of chromosome condensation that mimicked mitotic prophase but was followed by haphazard asymmetric segregation of chromatid clusters in abnormal metaphase/anaphase and premature reformation of nuclear membrane, resulting not in nuclear fragmentation, but in multiple micronuclei. This pattern of nuclear morphology was not observed in controls. The fraction of cells with micronuclear morphology increased with time in heated samples (from 3.6% at 4 hours to 16.6% at 16 hours), consistent with previous light microscopic analyses of nuclear fragmentation. Cells with multiple micronuclei subsequently exhibited features similar to necrotic cell death. Apoptosis was never observed. Moderate hyperthermia appears to induce a novel morphological pattern of cell injury and death in HeLa S3 cell lines that may be useful as a means of screening cell lines for nonmorphological analyses of hyperthermic injury.