The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2-3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2-3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphorylation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2-3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 microM and a Vmax of 0.1 nmol/min/oocyte. Co-injection of yeast hexokinase increased glucose phosphorylation by five-fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25-30-fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2-3H]glucose were analyzed by a multiple curve-fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate-limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three-fold lower extracellular concentration of 2-deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models.