Stimulation of nucleoside uptake in quiescent 3T3 cells by insulin and serum is preceded by a substantial lag phase. Our findings point to the length of the lag phase as a major target for regulation. The length of such phase varies markedly with the concentration of insulin (10(-9)-10(-6) M) or serum (0.5-10%) but it is not eliminated by high saturating levels of the activating agents. Further, variations in the temperature at which the stimulation process occurs (24-39 degrees C), addition of compounds like prostaglandin E1 (1-5 mug/ml) or theophylline (0.4 mM) and differences in the age of the cultures primarily affect the length of the lag time while the final uptake rates achieved are remarkably constant. Analysis of the temporal order of the events in the lag phase reveals that there is a discrete temperature-sensitive period located in the early and middle part of the lag, while the prostaglandin E1-sensitive step(s) appear to be toward the end of the lag. The transition from the basal to the stimulated rate of uptake is abrupt. Indeed, the kinetics of activation does not fit a sample exponential law but a high power of an exponential, suggesting that the switching mechanism involves cooperative steps. Since the transition is abrupt, the nucleoside uptake system exists largely in two alternative states either switched off or on. The regulation of the lag period is by the control of the time at which this switch occurs. On the basis of the data presented here, we propose a working hypothesis of uptake stimulation.