We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [14C] L-proline, [14C] L-alanine, and [14C] alpha-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (Kin) of 0.55 +/- 0.15 x 10(-4) ml/s/g and had no reversible compartment. AIB had a low Kin of 0.68 +/- 0.14 x 10(-4) ml/s/g and a significant reversible volume of 4.34 +/- 0.51 x 10(-3) ml/g in parietal cortex. L-alanine had the highest transfer coefficient, 3.11 +/- 0.26 x 10(-4) ml/s/g, and a reversible volume of 10.03 +/- 0.93 x 10(-3) ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport for L-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma and L-leucine and unchanged by sodium-free buffer, confirming its passage by the L1 system. L-alanine uptake was sodium-independent and not reduced by plasma. L-serine, L-cysteine, L-leucine and L-phenylalanine produced similar inhibition (66%) while L-alanine produced a lower inhibition (41%). L-arginine increased alanine uptake in cortex and thalamus. Adding L-serine to L-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest that L-alanine is transported by another L transport system different from the L1 system at the luminal membrane.