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.