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Objective: We have previously reported that low doses of [Met5]-enkephalin (YGGFM, met-enkephalin) and two of its derivatives (YGG and YG) enhanced and accelerated delayed-type hypersensitivity responses while much higher doses of these compounds suppressed these reactions. Since the underlying mechanisms by which this and other immunomodulatory effects occur have not been established, this report explores the in vitro modulation of Th1 and Th2 cytokine expression by these peptides. Methods: Murine splenocytes were stimulated with suboptimal concentrations of concanavalin A (ConA) in serum-free medium in the absence or presence of met-enkephalin, YGG, YG, [des-Tyr1]-met-enkephalin (GGFM), [D-Ala2], [D-Met5]-enkephalin or tyrosine (Y). Cell-conditioned supernatants were assayed for interferon-γ (IFN-γ), interleukin (IL)-2 and IL-4. Relative IFN-γ and IL-2 mRNA levels were assessed by reverse transcription-polymerase chain reaction. The enhancing and suppressive effects of met-enkephalin and YG on IFN-γ production were also tested in the presence of naloxone (Nx). Results: Met-enkephalin, YGG and YG modulated the in vitro production of IFN-γ in a biphasic manner: stimulation at low doses and inhibition at high doses. At higher concentrations, met-enkephalin and YG also suppressed the production of IL-2 (type 1) and IL-4, a type 2 cytokine. Nx reversed the enhancing effect of met-enkephalin on IFN-γ production without affecting its suppressive action or any of the immunomodulating effects of YG. The degradation-resistant analog [D-Ala2], [D-Met5]-enkephalin enhanced IFN-γ production but did not suppress it. Conclusions: YG, the minimal molecular requirement for enhancement and suppression of immune responses by these metabolites, appears to mediate exclusively an across-the-board suppression via low-affinity, nonclassical, nonopioid receptors.