Daily injections of 5 micrograms estradiol benzoate (EB) for 1, 2, 3, and 12 days decreased parametrial and retroperitoneal adipose tissue lipoprotein lipase activity in ovariectomized rats. Nearly all of this decrease occurred within the 1st day. In contrast, EB-induced body weight loss and hypophagia were very slight during the 1st day after EB administration and increased substantially over the next 2 days. EB did not affect gastrocnemius muscle lipoprotein lipase activity over the first 3 days, but did decrease muscle lipoprotein lipase activity after 12 days. These changes in adipose and muscle lipoprotein lipase activity suggested that EB-induced redistribution of fuel would be greater after 3 days of treatment than after 12 days of treatment. Tests with two different in vivo triglyceride uptake methods confirmed this suggestion. EB-treated rats were hypertriglyceridemic after 12 days of treatment, but not after 3 days of treatment. Elevated plasma triglycerides were associated with an increased rate of triglyceride entry. These findings are consistent with the hypothesis that EB administration temporarily results in redistribution of triglyceride fuel from adipose tissue to muscle, resulting in a transient decrease in rate of weight gain.