Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), we investigated the role of PPARalpha in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPARalpha (PPARalpha(-/-)), peroxisomal fatty acyl-CoA oxidase (AOX(-/-)), and in both PPARalpha and AOX (double knock-out (DKO)). Fasting for 48-72 h caused profound impairment of fatty acid oxidation in both PPARalpha(-/-) and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPARalpha(-/-) mice. The absence of PPARalpha in both PPARalpha(-/-) and DKO mice impairs the induction of mitochondrial beta-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal beta-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPARalpha and up-regulation of mitochondrial beta-oxidation and microsomal omega-oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPARalpha hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPARgamma and PPARdelta mRNA levels. PPARgamma and PPARdelta hepatic mRNA levels were lower in fed PPARalpha(-/-) and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPARgamma levels and a decrease in PPARdelta levels. Fasting did not change the PPAR isoform levels in AOX(-/-) mouse liver. These observations point to the critical importance of PPARalpha in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.