Transport of L-glutamate from blood to brain in equithesin-anesthetized rats was examined using in situ brain perfusion combined with multiple-time/graphical analysis. In situ perfusion allowed precise control of the composition of the perfusate, which was necessary for a detailed investigation of glutamate transport, while multiple time/graphical analysis permitted evaluation of the rapidly reversible volume and the period when the influx was unidirectional. Glutamate had no reversible volume and efflux from brain occurred after 30 s of perfusion. The in situ transfer coefficient (Kin) ranged from 0.74 +/- 0.07 mul/s per g in parietal cortex to 0.44 +/- 0.07 mul/s per g in hippocampus. L-Glutamate uptake was unaffected by removal of sodium from the perfusate, reduced by 5 mM L-glutamate, L-homocysteate, L-aspartate, plasma and 0.1 mM L-glutamate, while L-cystine did not reduce its uptake. These results suggest that the transport system for glutamate is saturated mainly by L-glutamate at physiological conditions and that it is not the sodium-independent x-C system since glutamate transport was not reduced by L-cystine except in hippocampus and that it was responsive to L-aspartate.