In order to examine the effect of dehydroepiandrosterone (DHEA) treatment on peroxisomal and mitochondrial β-oxidation capacity in muscle, brown adipose tissue (BAT), and liver, peroxisomal and mitochondrial palmitate oxidation capacity was measured in liver, BAT, heart, diaphragm, and skeletal muscles of rats fed an unrefined diet with or without added 0.61 DHEA acetate. After 6 weeks of treatment body weights of DHEA-treated rats were significantly lower than control rats. After 1 week of DHEA treatment the liver capacity for peroxisomal, and mitochondrial β-oxidation was markedly increased in DHEA-treated rats. Diaphragm capacity for peroxisomal, and mitochondrial β-oxidation was significantly increased in DHEAtreated rats after 2 weeks and 1 week, respectively. After 3 weeks of DHEA treatment the capacity of the gastrocnemius and soleus for mitochondrial β-oxidation was significantly increased in DHEA-treated rats. The BAT capacity for peroxisomal and mitochondrial β-oxidation was not affected by DHEA treatment. These results suggest that DHEA, at phamlacological levels, suppresses body weight gain and fat storage by increasing peroxisomal and mitochondrial β-oxidation capacity in the liver, diaphragm, and skeletal muscles.