Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.