Calcium-tolerant myocytes from the adult rat heart were used to study the effects of insulin on the kinetics of myocardial 3-0-methylglucose transport at 37 degrees C. Insulin increased the initial velocity of sugar influx without affecting the equilibrium uptake values. Maximal stimulation averaged 50-80%, with a half-maximal response at an insulin concentration of 0.1 nM and maximal stimulation occurring at 1 nM. The onset of insulin action was preceded by a lag-phase of 20 s, reaching maximal action by 60 s. The Vmax. of the glucose transport system was increased from 160 to 287 nmol/min per 10(6) cells with an unaltered affinity. Neither extracellular nor intracellular calcium was found to be involved in the stimulatory action of insulin. Removal of intracellular magnesium resulted in a loss of insulin action. This study demonstrates that activation of the cardiac glucose transporter by insulin is due exclusively to an increase in the maximal velocity representing one of the very early effects of insulin on myocardial metabolism. The data suggest involvement of magnesium in the transmission of the insulin signal.