Immunosuppressant FK506 is neuroprotective in experimental models of cerebral ischemia, but the molecular mechanisms underlying this neuroprotection remain unknown. We have demonstrated that FK506 inhibits the signaling pathways that regulate hypertrophic/proliferative responses in cultured astrocytes. Ischemia/reperfusion injury is associated with the proliferation and hypertrophy of astrocytes and with inflammatory responses. In the present work, we sought to determine whether FK506 neuroprotection after middle cerebral artery occlusion (MCAo) in rat is mediated via suppression of glia activation and changes in cytokine expression. Neurological deficits, infarct size, and astrocyte/microglial response were quantified in rats subjected to 90 min of MCAo. Changes in the mRNA expression of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in ipsilateral and contralateral cortices were determined by reverse transcription-polymerase chain reaction (RT-PCR). FK506 administered at 1 mg/kg, 60 min after MCAo, produced a significant improvement in neurological function and reduction of infarct volume. In FK506-treated rats, a significant reduction of IL-1beta, IL-6, and TNF-alpha expression was observed 12 h after reperfusion. FK506 neuroprotection was associated with a significant downregulation of IL-1beta expression in astrocytes and microglia in the injured side. FK506 selectively decreased the levels of TNF-alpha, and IL-1beta mRNAs in astrocytes in vitro, with no effect on transforming growth factor-beta 1 (TGF-beta1) and IL-6 expression. Moreover, FK506 inhibits lipopolysaccharide (LPS)-induced activation and cytokine expression in microglia in vitro. Our findings suggest that astrocytes and microglia are targets for FK506, and that modulation of glial response and inflammation may be a mechanism of FK506-mediated neuroprotection in ischemia.