In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound, suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell-type specific manner, leads to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro, cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occur in the insoluble fractions of cell homogenates, and that this in vitro activation can be attenuated by a soluble, suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100, or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurs in a narrow pH range (from pH 5.2 to pH 7.2), with a sharp increase in activation at the transition from pH 5.2 to pH 5.4, and can be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 µg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies, completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions, but not active proteases.