The development of diabetes in non-obese diabetic (NOD) mice, which normally takes between 3 and 7 months, can be accelerated by cyclophosphamide (CY) injections, with rapid progression to diabetes within only 2ยฑ3 weeks. This insulin-dependent diabetes mellitus (IDDM) can be prevented or delayed in CY-treated NOD mice by nicotinamide (NA). The present study was undertaken to determine the mode of cell death responsible for the development of IDDM in CYtreated male NOD mice and to investigate the effect of NA on beta-cell death. Apoptotic beta cells were present within the islets of Langerhans in haematoxylin and eosin-stained sections of the pancreata harvested from 3-and 12-week-old male NOD mice, from 8 h until 14 days after a single intraperitoneal injection of CY (150 mg/kg body weight). The maximum amount of betacell apoptosis in 3-week-old animals occurred 1ยฑ2 days after CY treatment (20 apoptotic cells per 100 islets), after which time levels of apoptosis declined steadily throughout the 14-day period studied. The incidence of beta-cell apoptosis in 12-week-old male NOD mice occurred in two peaks; the ยฎrst was recorded 8ยฑ24 h after CY treatment (30 apoptotic cells/100 islets), while the second, at 7 days (36 apoptotic cells per 100 islets), coincided with increased insulitis. Administration of NA 15 min before CY treatment, and thereafter daily, substantially reduced the amount of apoptosis and effectively eliminated (4 apoptotic cells per 100 islets) the second wave of beta-cell apoptosis seen at day 7 in 12-week-old animals given CY alone. These results show that apoptosis is the mode of beta-cell death responsible for the development of CY-induced IDDM and that prevention of IDDM by NA is associated with a reduction in beta-cell apoptosis.