Glutamate transporters may be important targets for anaesthetic action in the central nervous system. The authors investigated the effects of alphaxalone, an intravenous neurosteroid anaesthetic, on the activity of glutamate transporter type 3 (EAAT3). EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after applying L-glutamate (30 microM) in the presence or absence of alphaxalone. Responses were quantified by integrating current traces and are reported in microCoulombs (microC). Results are presented as means+/-S.E.M. L-Glutamate induced inward currents in EAAT3 expressing oocytes, and these currents were dose-dependently increased by alphaxalone. Alphaxalone at 0.01 to 3 microM significantly increased the inward currents. In addition, the treatment of oocytes with phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the transporter currents (1.0+/-0.2 to 1.4+/-0.2 microC; P<0.05). However, treatment with PMA plus alphaxalone did not increase responses further as compared with PMA or alphaxalone alone. Furthermore, pretreatment of oocytes with chelerythrine or staurosporine, two PKC inhibitors, did not affect basal transporter currents, but did significantly reduce alphaxalone-enhanced EAAT3 activity; whereas oocytes pretreated with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, showed significant reductions in basal and alphaxalone-enhanced EAAT3 activities. The above results suggest that alphaxalone enhances EAAT3 activity and that PKC and PI3K are involved in this effect.