Glial response to chronic morphine treatment was examined by immunohistochemistry of glial fibrillary acidic protein (GFAP), a specific marker for astroglial cells. Systemic administration of morphine (50 mg/kg, i.p.) once daily for 9 consecutive days led to significant increase in GFAP immunostaining density in the spinal cord, posterior cingulate cortex and hippocampus but not in the thalamus. This increase was attributed primarily to hypertrophy of astroglial cells rather than their proliferation or migration. When chronic morphine (20 microg/2 microl, i.t.) was delivered in combination with fluorocitrate (1 nmol/1 microl, i.t.), a specific and reversible inhibitor of glial cells, spinal tolerance to morphine analgesia was partly but significantly attenuated as measured by behavioural test and the increase in spinal GFAP immunostaining was also greatly blocked. The present investigation provides the first evidence for the role of glial cells in the development of morphine tolerance in vivo.