To test the hypothesis that clozapine-induced reduction of glutamate transporter-1 (GLT-1) expression is mediated by astrocytes, we studied the effects of clozapine on Glu transporters and Glu uptake in primary astrocyte cultures of the cerebral cortex. Astrocyte cultures treated for 48 h with clozapine exhibited a reduction in GLT-1 levels of about 50%, whereas glutamate-aspartate transporter (GLAST) levels remained unchanged. Glu uptake was also lowered, and this reduction was dose-dependent. Our findings indicate that clozapine reduces GLT-1 expression and function by a mechanism that directly involves astrocytes. A better understanding of the molecular events by which antipsychotics regulate Glu uptake can contribute to identify new targets for the treatment of schizophrenia.