A specific D-glucose-inhibitable [3 H] cytochalasin B-binding assay is used to examine the time course, reversibility, and hormone concentration dependency of an insulin-induced translocation of glucose transport systems from a large intracellular pool, associated with a low density subfraction of the microsomal membranes, to the plasma membrane in isolated rat adipose cells. The number of glucose transport systems in the plasma membrane and glucose transport activity in the intact cell are compared during the time course and reversal of insulin's action. Incubation of intact cells with 0.7 nM (100 microunits/ml) insulin at 37 C rapidly increases the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane fraction from 10 to 55 pmol and simultaneously decreases that in the low density microsomal membrane fraction from 95 to 34 pmol, each with a half-time of approximately 2.5 min. Furthermore, when maximally insulin-stimulated intact cells are exposed to a 300-fold excess of anti-insulin antibody, both effects of insulin are simultaneously reversed by 95% over a 30-min period with half-times of approximately 9 min. The concentrations of insulin producing half-maximal and maximal effects are approximately 0.11 nM (15 microunits/ml) and 0.35 nM (50 microunits/ml), respectively. The patterns of these reciprocal effects of insulin in time, during reversal, and with increasing hormone concentration are highly correlated (r= 0.93). At a steady state, the magnitude of insulin's stimulatory effect on the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane fraction closely correlates with the magnitude of insulin's stimulatory action on 3-O-methylglucose transport in the intact cell. However, during the onset of insulin's action, the appearance of cytochalasin B-binding sites in the plasma membrane fraction precedes the rise in 3-O-methylglucose transport in the intact cell (half-time of 4.0 min) by approximately 1.5 min. No such lag is observed during the reversal of insulin's action. These results directly demonstrate that insulin increases the number of glucose transport systems in the plasma membrane of the isolated rat adipose cell through a rapid and reversible translocation of glucose transport systems from a specific intracellular membrane pool. While this translocation of glucose transport systems to the plasma membrane fully accounts for the magnitude of insulin's stimulatory action on glucose transport activity in the intact cell, it appears to represent only one step in a multistep process.