Glutamate transporters play an important role in homeostasis of extracellular glutamate, a major excitatory neurotransmitter and a potential neurotoxin. In mammalian brain, glutamate transporter type 2 (EAAT2) is the most abundant form. Studies of molecular structures demonstrated that tyrosine 403 is critical in regulating the ion selectivity and transport mode of EAAT2. We hypothesized that wild type EAAT2 and its mutant at tyrosine 403 have different responses to volatile anesthetics, commonly used anesthetics that have been shown to affect glutamate transporter activity and decrease extracellular glutamate concentrations. We used site-directed mutagenesis and oocyte expression systems to test the hypothesis. Volatile anesthetics did not affect the activity of wild type EAAT2, isolated from rat hippocampus. When tyrosine 403 was replaced by histidine (Y403H), volatile anesthetics (isoflurane or halothane) at clinically relevant concentrations significantly decreased the transporter activity. Okadaic acid, a phosphatase inhibitor, significantly prolonged the isoflurane-induced inhibition. This inhibition was reversed by staurosporine and calphostin C, two protein kinase C (PKC) inhibitors, but not by the third PKC inhibitor, chelerythrine. Phorbol 12-myristate 13-acetate, a PKC activator, inhibited the activity of both wild type and Y403H EAAT2. This inhibition was also reversed by the same two PKC inhibitors but not by the third one. These results suggest that the switch of tyrosine 403 to histidine rendered EAAT2 sensitive to volatile anesthetics, a phenomenon that may require protein phosphorylation. PKC may be involved in the regulation of the activity of both wild type and Y403H EAAT2.