Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD x fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+-deprived granule neurons.