The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and beta-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [3H]U69,593, [3H]morphine, or 125I-beta-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor beta-endorphin binding. Cross-linking of 125I-beta-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both kappa- and mu-opioid receptors are expressed. There does not appear to be a significant difference in the level of mu nor kappa receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that kappa- and mu-opioid receptors are present in C6 glioma cells.