The purpose of this study was to determine whether subtilisin, a potent serine proteinase derived from Bacillus species contaminating smokeless tobacco, increases macromolecular efflux from the oral mucosa and, if so, whether local elaboration of bradykinin mediates this response. Using intravital microscopy, I found that suffusion of subtilisin elicits significant, concentration-dependent leaky site formation and an increase in the clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the in situ hamster cheek pouch (P < 0.05). Heat-inactivated subtilisin had no significant effects on macromolecular efflux. Subtilisin-induced responses were significantly attenuated by Hoe 140 and NPC 17647, two structurally distinct selective bradykinin B₂ receptor antagonists, but not by des-Arg⁹-[Leu⁸]bradykinin, a selective bradykinin B₁ receptor antagonist, or CP-96,345, a selective neurokinin-1 receptor antagonist. Aprotinin, but not leupeptin, significantly attenuated subtilisin-induced increase in macromolecular efflux. Indomethacin had no significant effects on subtilisin-induced responses. Collectively, these data indicate that subtilisin increases the macromolecular efflux from the in situ hamster cheek pouch in a catalytic-site-dependent fashion through local elaboration of bradykinin. This response does not involve the stimulation of local afferent nerves or the production of prostaglandins.