The expression of uncoupling protein (UCP)-3 mRNA in skeletal muscle is dramatically reduced during lactation in mice. The reduction in UCP-3 mRNA levels lowers the amount of the UCP-3 protein in skeletal muscle mitochondria during lactation. Spontaneous or abrupt weaning reverses the downregulation of the UCP-3 mRNA but not the reduction in UCP-3 protein levels. In lactating and virgin mice, however, fasting increases UCP-3 mRNA levels. Changes in UCP-3 mRNA occur in parallel with modifications in the levels of free fatty acids, which are reduced in lactation and are upregulated due to weaning or fasting. Modifications in the energy nutritional stress of lactating dams achieved by manipulating litter sizes do not influence UCP-3 mRNA levels in skeletal muscle. Conversely, when mice are fed a high-fat diet after parturition, the downregulation of UCP-3 mRNA and UCP-3 protein levels due to lactation is partially reversed, as is the reduction in serum free fatty acid levels. Treatment of lactating mice with a single injection of bezafibrate, an activator of the peroxisome proliferator-activated receptor (PPAR), raises UCP-3 mRNA in skeletal muscle to levels similar to those in virgin mice. 4-chloro-6-[(2,3-xylidine)-pirimidinylthio] acetic acid (WY-14,643), a specific ligand of the PPAR-alpha subtype, causes the most dramatic increase in UCP-3 mRNA, whereas troglitazone, a specific activator of PPAR-gamma, also significantly increases UCP-3 mRNA abundance in skeletal muscle of lactating mice. However, in virgin mice, bezafibrate and WY-14,643 do not significantly affect UCP-3 mRNA expression, whereas troglitazone is at least as effective as it is in lactating dams. It is proposed that the UCP-3 gene is regulated in skeletal muscle during lactation in response to changes in circulating free fatty acids by mechanisms involving activation of PPARs. The impaired expression of the UCP-3 gene is consistent with the involvement of UCP-3 gene regulation in the reduction of the use of fatty acids as fuel by the skeletal muscle and in impaired adaptative thermogenesis, both of which are major metabolic adaptations that occur during lactation.