Exposure of resting rat diaphragm for one hour in vitro to halothane (1-1.5, 2-2.5 and 4-4.5 per cent in oxygen) produced significant alterations of intracellular glucose disposition. Glycolysis (as measured by lactate production) increased, while glycogen formation was inhibited in a dose-related fashion. Net glucose uptake was unaffected by the anesthetic except during exposure to 4-4.5 per cent halothane, when 14 per cent depression of uptake was found. Total glycogen content decreased, due mainly to the inhibition of glycogen synthesis and to some extent to a stimulation of glycogenolysis. The anesthetic did not interfere with the effect of insulin on glucose uptake or the intracellular disposition of glucose. Creatine phosphate concentrations decreased following exposure of diaphragm to 1-1.5, 2-2.5 and 4-4.5 per cent halothane, while the adenosine triphosphate concentration declined after exposure to 4-4.5 per cent only. Although the mechanism(s) whereby halothane alters glucose and glycogen metabolism are unknown, it is possible that the anesthetic acts primarily by affecting membranes containing enzymes involved in the metabolism of glycogen.