Na(+)-K(+)-ATPase provides the driving force for cellular Na+ transport and exists in multiple isoforms that differ in ouabain sensitivities. We report that the Ki for ouabain inhibition of glucose-evoked short-circuit current, determined in intact rat ileal mucosa mounted in Ussing chambers, is higher in streptozocin-induced chronically diabetic rats than in age-matched controls. The changes in ouabain sensitivity seen in diabetes also occurred when intact ileum of age-matched controls was incubated in vitro with 2.8 x 10(-5) M glucagon for at least 80 min. The effect of glucagon was blocked by cycloheximide, indicating a role for protein synthesis. This suggests that changes in ouabain sensitivity seen in diabetes are produced by glucagon, the serum concentration of which increases in diabetes. Ouabain-dependent phosphorylation of Na(+)-K(+)-ATPase (backdoor phosphorylation) revealed a higher Km for phosphate in intestinal basolateral membranes obtained from diabetic rats compared with age-matched controls, again confirming a decrease in ouabain sensitivity. Furthermore, the mRNA encoding the alpha 1-isoform was upregulated 2.6-fold in chronically diabetic intestines. This suggests that the ouabain sensitivity seen during diabetes may be due to upregulation of the alpha 1-isoform, known to be less sensitive to ouabain than the other isoforms.