Our aims were to further characterize the stimulatory effect of glucagon on brown fat and to test the hypothesis that physiological levels of hyperglucagonemia would stimulate brown fat thermogenesis. In the first set of experiments, glucagon (1 mg/kg sc twice daily) or vehicle control was administered three times in 26 h. This large dose of glucagon produced increases in GDP binding to brown fat mitochondria. In addition, Scatchard analysis indicated a glucagon-induced increase in number of GDP binding sites without evidence for alteration in binding site affinity. No consistent increase in brown fat mitochondrial GDP binding was produced 2 h after a single injection of glucagon (1 mg/kg). In the second set of experiments, glucagon was administered intraperitoneally by constant osmotic minipump infusion. Glucagon in a dose of 150 micrograms.kg-1.day-1 for 5 days produced significant increases in GDP binding to brown fat mitochondria, whereas glucagon serum levels were increased but stayed within the usual physiological range. A larger dose of glucagon administered by constant infusion virtually eliminated body weight gain over 7 days while significantly increasing nucleotide binding (GDP) to brown fat mitochondria. An important role for glucagon in thermogenic regulation is suggested.