It is now widely accepted that insulin stimulation of glucose uptake by muscle cells is due to the activation of protein kinase B, leading to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane. Vanadate is a protein tyrosine phosphatase (PTP) inhibitor and a known insulin mimetic agent. Vanadate causes an increase of glucose transport in various tissues, but the mechanism of stimulation is not clearly understood. Hence in the present study, we have compared the mechanism of 2-deoxy-D-glucose transport induced by vanadate and insulin in isolated rat cardiomyocytes. Vanadate stimulated deoxyglucose transport in a time- and concentration-dependent manner. Insulin (100 nM) and vanadate (5 mM) stimulated 2-deoxy-D-glucose transport on an average by 3- and 2-fold respectively over basal values. The stimulation of glucose transport was accompanied by an activation of protein kinase B (PKB). This study also revealed that the activation of PKB and stimulation of 2-deoxyglucose uptake by vanadate and insulin are inhibited by treatment with wortmannin, a specific inhibitor of phoshatidylinositol 3-kinase (PI 3-kinase). Hence, we conclude that both insulin and vanadate follow the same signalling pathway downstream of PI 3-kinase to stimulate 2-deoxy-D-glucose transport.