Apart from impaired beta-oxidation, Pparalpha-deficient (Pparalpha(-/-)) mice suffer from hypoglycemia during prolonged fasting, suggesting alterations in hepatic glucose metabolism. We compared hepatic glucose metabolism in vivo in wild type (WT) and Pparalpha(-/-) mice after a short term fast, applying novel isotopic methods. After a 9-h fast, mice were infused with [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol for 6 h, and blood and urine was collected in timed intervals. Plasma glucose concentrations remained constant and were not different between the groups. Hepatic glycogen content was 69 +/- 11 and 90 +/- 31 microM/g liver after 15 h of fasting in WT and Pparalpha(-/-) mice, respectively. The gluconeogenic flux toward glucose 6-phosphate was not different between the groups (i.e. 157 +/- 9 and 153 +/- 9 microM/kg/min in WT and Pparalpha(-/-) mice, respectively). The gluconeogenic flux toward plasma glucose, however, was decreased in PPARalpha(-/-) mice (i.e. 142 +/- 9 versus 124 +/- 13 microM/kg/min) (p < 0.05), accounting for the observed decrease (-15%) in hepatic glucose production in Pparalpha(-/-) mice. Expression of the gene encoding glucose-6-phosphate hydrolase (G6ph) was lower in the PPARalpha(-/-) mice compared with WT mice. In conclusion, Pparalpha(-/-) mice were able to maintain a normal total gluconeogenic flux to glucose 6-phosphate during moderate fasting, despite their inability to up-regulate beta-oxidation. However, this gluconeogenic flux was directed more toward glycogen, leading to a decreased hepatic glucose output. This was associated with a down-regulation of the expression of G6ph in PPARalpha-deficient mice.