We investigated the role of Ca2⁢ and protein kinases/phosphatases in the stimulatory effect of insulin on glucose transport. In isolated rat adipocytes, the simple omission of CaCl2 from the incubation medium significantly reduced, but did not abolish, insulin-stimulated 2-deoxy glucose (2-DG) uptake. Pre-loading adipocytes with intracellular Ca2⁢ chelator, 5,5'-dimethyl bis (o-aminophenoxy)ethane-N,N,N'N' tetraacetic acetoxymethyl ester (5,5'-dimethyl BAPTA/AM) completely blocked the stimulation. Insulin raised intracellular Ca2⁢ concentration ([Ca2⁢]i) about 1.7 times the basal level of 72⁑5 nM, and 5,5'-dimethyl BAPTA/AM kept it constant at the basal level. This correlation between insulin-induced increases in 2-DG uptake and [Ca2⁢]i indicates that the elevation of [Ca2⁢]i may be prerequisite for the stimulation of glucose transport. Studies with inhibitors (ML-9, KN-62, cyclosporin A) of Ca2⁢-calmodulin dependent protein kinases/phosphatases also indicate an involvement of intracellular Ca2⁢. Additional studies with okadaic acid and calyculin A, protein phosphatase-1 (PP-1) and 2A (PP-2A) inhibitors, indicate an involvement of PP-1 in insulin action on 2-DG uptake. These results indicate an involvement of Ca2⁢-dependent signaling pathway in insulin action on glucose transport.