Anti-α-corticotropin [anti-ACTH(α)(1-13)] (also α-melanotropin) and anti-γ-endorphin antisera neutralized human leukocyte interferon activity but not fibroblast interferon activity. Human leukocyte interferon was not neutralized by anti-human lutenizing hormone (lutropin) or follicle-stimulating hormone (follitropin) antisera. Conversely, antisera to human leukocyte interferon neutralized ACTH activity. The neutralization of human leukocyte interferon by anti-human leukocyte interferon serum was partially blocked by ACTH. These studies show strong antigenic relatedness among human leukocyte interferon, ACTH, and endorphins, implying that there are underlying structural similarities. Structural relatedness is shown by pepsin cleavage of ACTH activity from human leukocyte interferon. The implications for the natural functions of human leukocyte interferon are discussed.