To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the α isoform of mouse PPAR (mPPARα) by homologous recombination. Mice homozygous for the mutation lack expression of mPPARα protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional activation of target genes were not observed. These results clearly demonstrate that mPPARα is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPARα-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.