Ionomycin is a Calcium ionophore and an antibiotic produced by Streptomyces conglobatus ATCC 31005. It is observed that LCLC 103H cells overexpressing the catalytic subunit of μ-calpain rapidly underwent cell death after treatment with ionomycin. The first signs of Ionomycin-induced cell death are detected 3 h after addition of the ionophore. Addition of 2 μM Ionomycin to LCLC 103H cells causes an instantaneous increase in intracellular Ca2+ concentration from 50 to 180 nM. Remarkably, calcium concentrations are raised transiently to 0.8-1.5 μM. DNA and protein analysis in Ionomycin-treated cultures revealed DNA fragmentation and PARP cleavage to an 85-kDa fragment typical of caspase-mediated apoptosis. Three hours after addition of ionomycin, the percentage of early apoptotic cells approximately doubles. Necrosis could be detected in ~1-5% of the Ionomycin treated cells as supported by simultaneously positive fluorescein labeling and propidium iodide uptake. Caspase activation in whole cells was followed by monitoring the increase in activity against Ac-DEVD-amc following Ionomycin treatment.