Clonidine and related imidazoline agents, beside binding to alpha 2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than alpha 2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine >>> epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displayed by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these "receptors" may influence astrocyte physiology by regulating the levels of GFAP.