CaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses. This study reports that the presence of extracellular Ca(2+) in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells. Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca(2+)-free buffers. When treatment buffers contained Ca(2+), CPE caused a rapid increase in CaCo-2 cell Ca(2+) levels, apparently because of increased Ca(2+) influx through a CPE pore. High CPE doses caused massive changes in cellular Ca(2+) levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca(2+) levels that appear responsible for activating apoptosis. Both CPE-induced apoptosis and oncosis were found to be calmodulin- and calpain-dependent processes. As Ca(2+) levels present in the intestinal lumen resemble those of Ca(2+)-containing treatment buffers used in this study, perturbations in cellular Ca(2+) levels and calpain/calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.