WY-14,643, a lipid-lowering drug, increases basal rates of oxygen uptake in perfused livers. Because peroxisomes consume oxygen for H2O2 production and are induced by WY-14,643 treatment, it is possible that peroxisomal beta-oxidation can account for some of this increase in cellular respiration. Therefore, cyanide, an inhibitor of mitochondrial cytochrome oxidase, was infused into livers of WY-14,643-fed rats (0.1% WY-14,643 in laboratory rat chow for 1, 21, and 105 days) to assess peroxisomal cyanide-insensitive respiration. As expected, the addition of cyanide abolished oxygen uptake nearly completely; however, after approximately 20 min oxygen consumption unexpectedly returned to basal levels in 105-day WY-14,643-treated animals but not in untreated controls. Urea synthesis, a process dependent upon ATP, was decreased and remained low during cyanide infusion in livers from both groups, indicating that mitochondria were not responsible for this unusual increase in oxygen uptake in the presence of cyanide. Methanol metabolism, which requires oxygen to form H2O2, was decreased from 37 +/- 5 to 6 +/- 1 micromol/g/hr in all groups treated with cyanide; however, it was increased significantly about 20 min later to 25 micromol/g/hr in livers from WY-14,643-treated rats, indicating that oxygen for peroxisomal H2O2 production is involved in cellular respiration in the presence of cyanide. Fasting abolished the recovery of both oxygen uptake and methanol metabolism in WY-14,643-fed rats, suggesting that ATP for acyl CoA synthetase, an enzyme which metabolizes fatty acids to acyl CoA compounds, is provided by glycolysis. Indeed, oleate significantly increased methanol metabolism in fed control rats from 8 +/- 4 to 26 +/- 3 micromol/g/hr in the presence of cyanide, indicating that fatty acid supply is necessary for peroxisomal respiration. Taken together, these experiments demonstrate that when mitochondrial respiration is inhibited, livers from rats fed WY-14,643 chronically have the unique ability of metabolizing fatty acids through the peroxisome using glycolytic ATP.