High affinity binding sites for β-endorphin_1–31 (β-EP) have been observed on transformed mononuclear cells such as the human U937 monocyte-like cell line and the murine EL4-thymoma line, and on normal murine splenocytes. Binding of β-EP at these sites is resistant to competition by naloxone and other opiate receptor ligands but sensitive to N-acetyl-β-endorphin_1–31 (N-Ac), cations and GTP-γ-sulfate. Thus, the following studies were done to determine the functional significance of binding β-EP and N-Ac. β-EP suppressed phytohemagglutinin (PHA)-stimulated [³H]thymidine uptake in a dose-dependent, naloxoneinsensitive fashion. β-Endorphin_1–27, (des)-tyrosine β-endorphin_2–31, or N-Ac failed to duplicate the suppressive effect of β-EP. However, N-Ac, which is equipotent to β-EP at displacing ¹²⁵I-β-EP bound to murine splenocytes or U937 cells, antagonized the suppressive effect of β-EP. Taken together with previous binding studies, the present observations suggest that β-EP effects receptormediated responses on normal immunocytes that do not depend on the activation of neuronal-like opiate receptors which are naloxone-sensitive. N-Ac, which shows minimal binding to such brain opiate receptors, is a potent functional antagonist of the naloxone-insensitive immunocyte receptor for β-EP.