Diazepam-binding inhibitor has been initially isolated from the rat brain from its ability to compete with benzodiazepines for their receptors. We have recently shown that the octadecaneuropeptide (diazepam-binding inhibitor-(33–50) or ODN) induces an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in astroglial cells. The purpose of the present study was to determine whether central-type benzodiazepine receptors or peripheral-type benzodiazepine receptors are involved in the response of cultured rat astrocytes to ODN. The mixed central-/peripheral-type benzodiazepine receptor ligand flunitrazepam (10⁻¹⁰ to 10⁻⁶ M), the specific peripheral-type benzodiazepine receptor agonist Ro5-4864 (10⁻¹⁰ to 10⁻⁶ M) and the peripheral-type benzodiazepine receptor `antagonist' PK 11195 (10⁻⁹ to 10⁻⁶ M) all induced a dose-dependent increase in [Ca²⁺]_i. At high doses (10⁻⁷ to 10⁻⁵ M), the central-type benzodiazepine receptor agonist clonazepam also mimicked the stimulatory effect of ODN on [Ca²⁺]_i. However, the [Ca²⁺]_i rise induced by ODN was blocked neither by PK 11195 nor by the central-type benzodiazepine receptor antagonist flumazenil (10⁻⁶ M each). Binding of [³H]flunitrazepam to intact astrocytes was displaced by low concentrations of the peripheral-type benzodiazepine receptor ligands flunitrazepam, Ro5-4864 and PK 11195, and by high concentrations of clonazepam. In contrast, ODN did not compete for [³H]flunitrazepam binding in intact cells. These data indicate that the effect of ODN on Ca²⁺ mobilization in rat astrocytes is mediated by high affinity receptors which are not related to classical benzodiazepine receptors. ©1997 Elsevier Science B.V. All rights reserved.