Previous studies have shown that genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis are downregulated in liver and skeletal muscle of rats during lactation. However, biochemical mechanisms underlying these important metabolic adaptations during lactation have not yet been elucidated. As all these genes are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (Pparalpha), we hypothesized that their downregulation is mediated by a suppression of Pparalpha during lactation. In order to investigate this hypothesis, we performed an experiment with lactating and nonlactating Pparalpha knockout and corresponding wild-type mice. In wild-type mice, lactation led to a considerable downregulation of Pparalpha, Ppar coactivators Pgc1alpha and Pgc1beta, and Pparalpha target genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis in liver and skeletal muscle (P<0.05). Pparalpha knockout mice had generally a lower expression of all these Pparalpha target genes in liver and skeletal muscle. However, in those mice, lactation did not lower the expression of genes involved in fatty acid utilization and thermogenesis in liver and skeletal muscle. Expression levels of Pparalpha target genes in lactating wild-type mice were similar than in lactating or nonlactating Pparalpha knockout mice. In conclusion, the present findings suggest that downregulation of Pparalpha and its coactivators in tissues with high rates of fatty acid catabolism is responsible for the reduced utilization of fatty acids in liver and skeletal muscle and the reduced thermogenesis occurring in the lactating animal, which aim to conserve energy and metabolic substrates for milk production in the mammary gland.