The polypeptide β-endorphin binds to cultured bovine adrenal cortical cells in a naloxone insensitive manner, β-endorphin and N-Acetyl-β-endorphin are equipotent in inhibiting binding. The amino terminal 27 amino acid fragment referred to as β-endorphin[1-27] shows no ability to inhibit binding, whereas the carboxy-terminal tetrapeptide Lys-Lys-Gly-Glu partially inhibits binding. ACTH, angiotensin II and met-enkephalin show little or no ability to inhibit β-endorphin binding. Competition bin-ding reveals an apparently single affinity class with Kd of 33 nM. Molecular cross linking experiments reveal putative receptor subunits of 85 kD, 64 kD, 54 kD and 44 kD. The lower molecular weight bands are preferentially cross-linked by a hydrophobic cross linking reagent, in contrast to the two higher molecular weight bands, which are cross linked equally by hydrophobic and water soluble cross linking reagents. The β-endorphin binding characteristics of adrenal cortical cells revealed here are quite similar to those of a class of non-opioid β-endorphin receptors previously shown to exist in cells of the immune system.