Tonin was isolated from rat submandibular glands by a very convenient procedure consisting of sequential anion-exchange, hydrophobic interaction and gel filtration chromatographies. The method is superior to earlier purifications as it consists of fewer stages, resulting in a much higher recovery (41%) of tonin. The final preparation was seen to be pure on SDS gel electrophoresis (M(r) 32,800) and on gel isoelectric focusing (pI 6.15). The stability of tonin and its interaction with various inhibitors were investigated, and compared with the corresponding behavior of rat tissue kallikrein.