The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that modulate lipid and glucose homeostasis. In the clinic, PPARalpha and PPARgamma agonists are used to treat hypertriglyceridemia and insulin resistance of diabetes, respectively. To gain further insight into the molecular mechanisms underlying the therapeutic actions of these drugs, we have by two-dimensional electrophoresis and mass spectrometry performed a comparative analysis of the hepatic protein expression profiles of lean and obese (ob/ob) mice, and obese mice treated with WY14643 (PPARalpha agonist) or rosiglitazone (PPARgamma agonist). We found that livers from obese mice displayed higher levels of enzymes involved in fatty acid oxidation and lipogenesis compared to lean mice and these differences were further amplified by treatment with both PPAR activators. WY14643 normalized the expression levels of several enzymes involved in glycolysis, gluconeogenesis and amino acid metabolism in the obese mice to the levels of lean mice, whereas rosiglitazone partially normalized levels of enzymes involved in amino acid metabolism. In summary, a classical proteomics approach was successfully used to characterize differences at the hepatic proteome level between lean and obese diabetic mice, to map metabolic pathways affected by treatment, and to discriminate between effects caused by treatment with agonists of the closely related PPARalpha and PPARgamma receptors.