The high-affinity muscarinic antagonist L-[3H]quinuclidinyl benzi-late (L-[3H]QNB) was used to label muscarinic acetylcholine receptors in the astrocyte-enriched fraction isolated from rat hippocampus. Analysis of the saturation and drug inhibition data showed that [3H]QNB binding in the astroglia fraction was saturable, of high affinity (Kd = 0.026 nM) and competitively inhibited by muscarinic antagonists and agonists. The Kd for [3H]QNB binding sites in the astroglia fraction was similar to that found for muscarinic receptors in the hippocampal homogenate and in the hippocampal synaptic plasma membranes. The density of [3H]QNB binding sites in the bulk-isolated astroglia fraction (Bmax =0.852 pmol/mg protein) was, however, much lower than that in the hippocampal synaptic plasma membranes (Bmax equal to about 2 pmol/mg protein). The results suggest that hippocampal astrocytes possess specific [3H]QNB binding sites with receptor binding characteristics of neuronal muscarinic acetylcholine receptor.