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Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of prokallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SOS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.