We observed previously that lipopolysaccharide (LPS) 18 h after i.p. injection of guinea pigs increased transepithelial potential difference (Vt), hyperpolarization responses to methacholine, and hyperosmolarity-induced, epithelium-derived relaxing factor (EpDRF)-mediated relaxation responses, in excised and perfused tracheal segments. To investigate their roles in these changes, the effects of cytokines on in vitro epithelial bioelectric and smooth muscle mechanical responses were investigated using the isolated, perfused trachea preparation. Tracheas were incubated (6 h) with LPS or IL-1β, IL-4, IL-13, IFN-γ, TNF-α, singly or in combination. Incubation with LPS and cytomix (IL-1β + IFN-γ + TNF-α together) had no effect on muscle reactivity to methacholine, but potentiated D-mannitol-induced relaxation. Individually, IL-1β and IFN-γ inhibited methacholine-induced contractions and potentiated D-mannitol-induced relaxation responses. TNF-α increased contractions to methacholine but had no effect on relaxation responses to D-mannitol. Methacholine elicited hyperpolarization in low concentrations and depolarization in high concentrations. The individual cytokines decreased the hyperpolarization response to low methacholine concentrations and increased the depolarization response to high methacholine concentrations but had no effect on Vt responses to D-mannitol. Cytomix did not affect Vt responses to methacholine, but potentiated both the hyperpolarization and depolarization responses to D-mannitol. In Ussing chambers all agents except IL-1β and IFN-γ increased Vt; IL-1β decreased slightly but none of the other agents affected transepithelial resistance (Rt). The results indicate that cytokines and LPS alter smooth muscle reactivity to methacholine, potentiate EpDRF-mediated relaxation responses and, thereby, mimic the effects of LPS treatment in vivo, but do not recapitulate LPS' effects on Vt responses.