Previous studies in this laboratory demonstrated that insulin, when administered simultaneously with glucosamine, significantly lowered the hyperglycemic effect of the latter. This result conflicts with reports suggesting that glucosamine competes with glucose at the site of insulin action. To explore further this apparent contradiction, the effect of insulin and glucosamine on glucose metabolism by the intact rat diaphragm preparation was studied. The distribution ratio of glucose-1-14C and glucosamine-1-14C, the glycogen content, and the 14CO2 production were measured in the presence or absence of insulin after 90 and 120 min incubation. Insulin increased glucose and glucosamine penetration 7 and 4 times respectively. Glucosamine did not interfere with glucose entry into the cell either in the presence or absence of insulin. Glycogen synthesis was not impaired by glucosamine, although glucosamine itself did not contribute to glycogen formation. 14CO2 production from glucose-1-14C was markedly depressed by glucosamine, and glucosamine-1-14C did not contribute to 14CO2 production. These results indicate that in muscle tissue, glucosamine interferes neither with the glucose transport across the cell membrane, nor with the metabolic pathway of glucose to glycogen; but glucosamine, or a metabolic intermediate, does inhibit some metabolic step in the breakdown of glucose to CO2.