The in vitro and in vivo effect of aliphatic diketones has been studied on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) D,L-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating EC 220.127.116.11 activity). Neurotoxic diketone, 2,5-hexanedione (2,5-HD), but not 2,4-hexanedione (2,4-HD), a non-neurotoxic diketone, inhibited GAPDH in rat bran homogenate preincubated with 25 mM diketones for 20 min. If the preincubation period was increased to 2 h, approximately 25% and 55% inhibition of GAPDH activity was observed with 1 mM and 5 mM 2,5-HD respectively. The inhibition of GAPDH activity was also seen in sciatic nerves but not in the brain or liver homogenates of rats chronically intoxicated with 2,5-HD for 12 weeks. The inhibition of GAPDH by 2,5-HD appears to be selective, and thus confirms earlier data from this laboratory.