We investigated the effect of several opioid peptides on the activation of murine peritoneal exudate macrophages (M phi) in vitro. M phi were treated with interferon (IFN) as a priming agent and bacterial lipopolysaccharide (LPS) as a triggering agent in the presence or absence of opioid peptides. M phi activation was assessed by their tumoricidal activity. When treatment with IFN and LPS resulted in a high level activation of M phi, dynorphin-A exerted no further enhancing effect. When treatment induced only weak activation, however, dynorphin-A augmented the M phi activation. Leucine-enkephalin, methionine-enkephalin, and also beta-endorphin had augmenting effects. An opioid receptor antagonist, naloxone, reduced the effect of dynorphin-A and beta-endorphin. When M phi were treated sequentially with IFN and LPS, beta-endorphin operated in combination with LPS only. Moreover, beta-endorphin was effective for already activated M phi. These results indicate that opioid peptides act on M phi via classical opioid receptors, and that responsiveness to opioid peptides is induced in the triggering stage of M phi activation.