Two immortalized cell lines, sup (+) and sup (), derived from mutagenized Syrian hamster embryo cells, were used to study the relationship and temporal order between calcium and ceramide signals during apoptosis. The early preneoplastic cells, termed sup (+), suppress tumorigenicity when hybridized with tumor cells, whereas later-stage sup () cells do not. In reduced serum conditions, sup (+) cells cease proliferating and undergo apoptosis; in contrast, sup () cells continue slow growth and undergo necrosis. In sup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum treatment of sup (+) cells but are not found in sup () cells. Inhibiting ER calcium depletion in low serum-treated sup (+) cells by treating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup () cells by treating with low serum plus thapsigargin results in elevated ceramide levels and apoptosis. Furthermore, C₆-ceramide treatment induced apoptosis of sup () cells in low serum, a condition that does not normally cause apoptosis. C₆-ceramide treatment did not induce apoptosis in either sup (+) or sup () cells in 10% serum but did cause G₂/M arrest. These studies show that ceramide production is downstream of ER calcium release.