1. The interaction of L-cysteine with three excitatory amino acid transporter subtypes cloned from human brain (EAAT1-3) was examined by measuring transporter-mediated electrical currents and radiolabelled amino acid flux in voltage-clamped Xenopus oocytes expressing the transporters. 2. L-Cysteine was transported by the neuronal subtype EAAT3 (EAAC1) with an affinity constant of 190 microM and a maximal rate of flux similar to that of L-glutamate; the relative efficacies (Vmax/K(m)) of the EAAT1 and EAAT2 subtypes for transporting L-cysteine were 10- to 20-fold lower. 3. Changing the ionization state of L-cysteine by raising the external pH did not significantly change the apparent affinity, transport rate, or magnitude of currents induced by L-cysteine, suggesting that both the neutral zwitterionic and anionic forms of the amino acid are transported with the same net charge stoichiometry. 4. In addition to competing with L-glutamate for uptake by the neuronal carrier, L-cysteine caused transporter-mediated release of transmitter by heteroexchange; both actions would elevate extracellular glutamate concentrations and may thus contribute to the known excitotoxic actions of L-cysteine in the brain. 5. Because the EAAT3 transporter is also expressed in tissues including kidney and intestine, the results suggest the possibility of a heretofore unrecognized mechanism of L-cysteine uptake in peripheral tissues as well as in brain.