The cytotoxicity and its underlying mechanisms induced by gliotoxin (GT), an immunosuppressive agent, in macrophages are poorly understood. We report here that GT induced a rapid apoptosis (DNA fragmentation and hypodiploid nuclei obtained within 4 hrs of treatment) in murine macrophages PU5-1.8 in a dose-dependent and cell cycle-independent manner. The GT-induced apoptosis was suppressed by z-Asp, z-VAD-fmk and antioxidants suggesting that production of reactive oxygen species (ROS) and activation of caspases were important in this process. Also, release of cytochrome c from mitochondria was found to be an early event (within 1 hr) after addition of GT (250 ng/ml) and its presence in the cytosol was sufficient to elicit apoptosis. Interestingly, the release of cytochrome c was not accompanied by a reduction in the mitochondrial membrane potential (psi m) as determined by several psi m-sensitive fluorescent indicators. Taken together, our results indicate that GT is a potent apoptotic agent in PU5-1.8 cells and the loss of psi m is not a universal early marker for apoptosis.