The role of insulin receptor occupancy in the stimulation of glucose transport has been studied in isolated rat adipocytes. At 37 degrees C, under steady-state conditions, the time needed to fill the fraction of receptors (less than 1%) required for an initial measurable effect varied with insulin concentration from less than 10 s at 100 ng/mL to 90 s at 0.5 ng/mL. However, at all insulin concentrations there was an initial lag period before any activation was seen. The length of the initial lag was inversely related to the insulin concentration, lasting 2 min at 0.5 ng/mL and only 30-40 s at 5-500 ng/mL (maximal levels). A similar discrepancy was noted between dissociation of prebound insulin and the loss of insulin's effects on transport. At an insulin concentration of 0.3 ng/mL, half of the insulin effect was lost within 12 min; the t1/2 of dissociation was 8 min. When the insulin concentration was increased to 10 ng/mL, the t1/2 of dissociation increased only to 10 min while the t1/2 of deactivation was now 60 min. In conclusion, (1) kinetic studies reveal a time-requiring step between insulin binding and early effects on glucose transport, (2) a low level of insulin binding (less than 1% occupancy) is all that is necessary to initiate the insulin stimulus-response sequence, and (3) the rate of deactivation is closely related to the steady-state level of insulin binding, and with increasing insulin concentrations this rate slows and diverges from the rate of dissociation of insulin from receptors.