The abilities of insulin and the insulin mimickers spermine and H,O, to stimulate 3-0-methyl glucose transport in isolated rat fat cells were studied in an attempt to determine possible common mechanisms of action. All three agents caused a seven-to 12-fold stimulation of initial rates of glucose transport with insulin being the most effective agent. Insulin and spermine displayed similar time courses for the onset of their stimulation of transport; an initial lag before any effect was seen and then a gradual rise until the full effect was reached. The time course of H,O, activation of glucose transport was different since stimulation was seen at the earliest time point tested and then gradually rose to the maximal effect. Trypsinization of cells removed insulin receptors and rendered the cells insensitive to insulin but not to spermine or H,O,. However, trypsinization did alter the time course of H,O, action, causing an initial lag phase to appear and a general slowing of the activation kinetics. Pretreatment of cells with 2,4-dinitrophenol to lower ATP level, prevented the stimulatory effects of insulin and the mimickers. AII three of the agents revealed a similar temperature dependency for stimulated glucose transport, resulting i n linear Arrhenius plots with activation energies of 10.2-11.4 kcalimole. These results show that (1) H,O, does not act directly on the glucose transport system of rat adipocytes and (2) insulin and H,O most likely act through a common energy-dependent biochemical pathway to stimulate glucose transport, but H,O, enters the stimulus-response sequence distal to the initial steps in insulin action.