Expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) has been shown in liver of chicks, but effects of its activation have not yet been investigated. In this study, laying hens were treated with clofibrate, a synthetic PPARalpha agonist, to investigate the effects of PPARalpha activation on liver lipid metabolism. Hens receiving a diet containing 5 g of clofibrate/kg had a lower food intake and higher liver mRNA concentrations of typical PPARalpha target genes (carnitine palmitoyltransferase 1A, acyl-coenzyme A oxidase, bifunctional enzyme, lipoprotein lipase) involved in hepatic mitochondrial and peroxisomal beta-oxidation and plasma triglyceride clearance than control hens that received the same diet without clofibrate (P<0.05). Hens treated with clofibrate also had lower mRNA concentrations of fatty acid synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and low-density lipoprotein receptor, proteins involved in fatty acid biosynthesis and cholesterol biosynthesis and uptake, than hens fed the control diet (P<0.05). These changes in clofibrate-treated hens were accompanied by reduced liver triglyceride concentrations, strongly diminished very low density triglyceride and cholesterol concentrations (P<0.05), a disturbed maturation of egg follicles, a complete stop of egg production, and a markedly reduced plasma 17-beta-estradiol concentration (P<0.05). In conclusion, it is shown that clofibrate has complex effects on hepatic lipid metabolism in laying hens that mimic PPARalpha activation in mammals, affect maturation of egg follicles, and lead to a stop of egg production. Because clofibrate treatment strongly reduced food intake in the hens, some of these effects (i.e., egg production) may have been due to a low energy and nutrient intake.