Objective: Evidence indicates that estrogen depresses hepatic lipid oxidation. We tested the hypothesis that estradiol (E2) treatment depresses transcription of carnitine palmitoyltransferase-1 (Cpt 1) mRNA and increases adiposity. Research Methods and Procedures: Six ovariectomized female rats were given a subcutaneous pellet of E2 (5 mg/d), and six were given placebo. Rats were pair-fed by group for 18 days. Body composition was assessed chemically: mRNA for liver Cpt 1, adipose tissue uncoupling protein-2 (Ucp 2), and quadriceps Ucp 3 by Northern analysis; serum glucose, triglycerides (TGs), and free fatty acids by standard techniques; and serum insulin and glucagon by radioimmunoassay. Results: E2-treated rats lost more weight than placebo-treated rats (37.3 ± 6.0 vs. 16.2 ± 2.6 g, p < 0.01), but did not differ in final carcass composition (adjusted for eviscerated body mass). E2-treated rats had lower liver Cpt 1 (p < 0.001) and skeletal muscle Ucp 3 (p < 0.05) mRNA and lower concentrations of glucose, glucagon, and free fatty acids (p < 0.05). E2-treated rats tended to have higher insulin (p = -0.067) and TG (p = 0.097). TG tended to be correlated with Cpt 1 mRNA (r = -0.56 and p = 0.07). Discussion: These results suggest that, although E2 is likely to suppress lipid oxidation and promote TG synthesis, these effects are not manifested in a relative increase in carcass adiposity after 18 days of treatment, at least under conditions of negative energy balance. The possible role of E2-mediated changes in insulin and glucagon secretion on hepatic substrate metabolism warrants further study.