We determined the ability of urinary trypsin inhibitor (UTI), which is a Kunitz-type protease inhibitor present in serum and in urine, to inhibit rat peritoneal mast cell (RPMC) mediator release induced by several stimuli. UTI attenuated the immunoglobulin E-mediated release of both preformed(histamine) and newly formed (leukotriene C4) mediators from RPMCs. Inhibition (21% ± 5%) of the anti-IgE-triggered release of histamine was observed after a 30-minute incubation of RPMCs with UTI (5 µmol/L). To investigate the specificity of the UTI effect, we studied the stimulatory activity of phorbol ester (phorbol 12-myristate 13-acetate (PMA)) or calcium ionophore A23187 in control and UTI-treated mast cells. The efficacy of UTI as an inhibitor was dependent on the nature of the stimulus, because histamine release induced by PMA-mediated or calcium ionophore A23187-mediated processes was not inhibited by UTI. A series of structurally distinct protease inhibitors did not inhibit IgE-induced release of mediators from RPMCs. The Kunitz-type protease inhibitors are important in the regulation of RPMC function. In parallel with the UTI-related decrease in anti-IgE stimulatory activity on mediator release, increased microviscosity of membrane lipids could be observed by two independent experiments on fluorescence polarization with diphenylhexatriene (DPH) and on the fluorescence probe fluorescein isothiocyanate-concanavalin A. UTI reduces mediator release by a mechanism-possibly an interruption of the coupling of receptor and effector systems-because UTI acts as an agent to decrease biologic lipid membrane fluidity.