Opioid receptors were characterized in glial and neuronal homogeneous cultures of embryonic chick forebrain, using [3H]naloxone as a labelled ligand. Binding experiments were performed on intact cells. The specific binding of [3H]naloxone reached equilibrium after 1 min. The apparent dissociation constants were estimated as 0.51 nM for glial and 0.63 nM for neuronal cells. Equilibrium measurements indicated the apparent positive cooperativity of the binding, resulting in Hill coefficients of 2.61 for glial and 2.04 for neuronal cells. Competition of unlabelled naloxone for specific binding sites resulted in maximum-shape curves in glial cells if measured at low receptor occupancy. This supports the positive cooperativity of ligand binding. Opioid agonists, ethylketocyclazocine (EKC), morphine and [D-Ala2,L-Leu5]enkephalin (DALA), provoked biphasic competition curves in both cell types with a characteristic maximum at low competitor concentrations. The possible physiological role of glial opioid receptors in neuron-glia communication and the significance of cooperativity is discussed.