The in vitro uptake of [35S] cysteine was studied in crude synaptosomal preparation of the cerebral cortex of rat. The accumulation of cysteine was found to be temperature- and time-dependent. It was linear at least for four minutes at 37 C with characteristics of saturable kinetics. Uptake of cysteine was Na+- and K+-dependent. Increasing the Na+ ion concentration increased the accumulation of cysteine in synaptosomal preparations; unlike the Na+ ion, an increase was accumulated against concentration gradients by a saturable mechanism. Double reciprocal plot of the cysteine uptake suggests two types of affinity systems, with Km values for the high-affinity uptake of about 12.2 microM and for the low-affinity uptake of about 4 mM. The high-affinity uptake was also significantly inhibited by ouabain, a potent inhibitor of the Na+-K+-dependent ATPase, and other metabolic inhibitors. The results of the effects of cysteine analogues and uptake also suggested that it is a substrate-specific high-affinity uptake system for cysteine.