Asthma is characterized by bronchial inflammation and hyperresponsiveness that involves mast cell tryptase and potentially its specific receptor PAR-2 (protease activated receptor 2). Tryptase increases free intracellular calcium concentration ([Ca²⁺]_i), a key step in human airway smooth muscle cells (HASMC) activation. The aim of the study was to analyze the effect of PAR-2 gene silencing on HASMC, in terms of calcium response, since no antagonist is available for this receptor. Five siRNA against PAR-2 were synthesized and transfected in HASMC using lipid agents and PAR-2 expression was examined using western blot, FACS, immunocytochemistry and RT-PCR. [Ca²⁺]_i was measured using microspectrofluorimetry in response to tryptase, the activating peptide SLIGKV, trypsin or caffeine. Two siRNA significantly inhibited PAR-2 expression in terms of both total and surface protein expression, as well as mRNA levels. Tryptase- and SLIGKV-induced transient increase in [Ca²⁺]_i was significantly inhibited after transfection with the most appropriate siRNA, whereas neither trypsin nor caffeine response was altered. Two control siRNA had no effect in terms of both PAR-2 expression and calcium response. Transfection efficiency was maximal after 24h and disappeared after 48h. Gene silencing using siRNA can thus be used in vitro to assess the function of PAR-2 in HASMC.