Brain capillaries isolated from 2-month-old male and female Sprague-Dawley rats were used to study the transport of neutral amino acids. The uptake of alanine, leucine, and alpha-methylaminoisobutyric acid (MeAIB) was a linear function of time for the first minute of incubation. Based on these observations, an incubation time of 1 min was used to measure transport activities. The intracellular water volume of the isolated capillaries was 2.2 microliters/mg protein. This value was significantly lower (1.8 microliter/mg protein) when measured in the absence of sodium. L-Alanine, L-serine, and L-cysteine were taken up from the abluminal surface of brain capillaries by a sodium- and energy-dependent, carrier-mediated system. This uptake, for the most part, was not inhibited by MeAIB. System ASC (alanine-serine-cysteine) appeared to be of primary importance for the transport of these amino acids in isolated brain capillaries. The apparent Km and Vmax values for L-alanine uptake by ASC transport, based on the Hofstee plot presentation, were 1.3 mM and 0.975 nmol/microliter water content/min, respectively. The results also indicate that the transport of MeAIB and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) was limited to the sodium-dependent system A (alanine) and the sodium-independent system L (leucine), respectively.