A monensin-resistant mutant Monr-31, derived from Chinese hamster ovary (CHO) cell line, has been shown to have a reduced number of insulin receptors and a reduction in glucose uptake in response to insulin. We have further investigated the possibility that altered glucose uptake in Monr-31 cells is related to an alteration in the activity of the insulin receptor. Uptake of glucosamine, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose in Monr-31 cells was one-half to one-third that of CHO cells. The cellular content of the glucose transporter in Monr-31 was reduced to about one-third that of CHO as assayed by use of an antiglucose transporter antibody. After transfection with the human insulin receptor cDNA, we obtained clones CIR-0 from CHO, and MIR-2 and MIR-15 from Monr-31; CIR-0 expressed a tenfold higher level of the insulin-binding activity than did CHO, and MIR-2 and MIR-15 expressed a 20-fold higher level than did Monr-31. Glucose uptake in both CHO and CIR-0 was significantly enhanced by exogenous insulin, but not in Monr-31, MIR-2, and MIR-15. The beta-subunits of insulin receptor in CHO, CIR-0, Monr-31, and MIR-2 were similarly phosphorylated. The decreased glucose transport activity in Monr-31 cells is discussed in relation to the absence or presence of insulin receptor expression.