A basolateral membrane (BLM) enriched fraction of the homogenized rat kidney contained kallikrein and prekallikrein which differ from urinary kallikrein. Triton X-100 (0.1%) or melittin (10(-7) - 10(-5)M) solubilized the membrane-bound enzyme. Prekallikrein was activated by trypsin and plasmin. Active kallikrein and activated prekallikrein cleaved the chromogenic substrate S-2266 and released bradykinin from kininogen. Aprotinin and antiserum to rat urinary kallikrein inhibited BLM kallikrein. Gel electrophoresis separated activated BLM prekallikrein and kallikrein; prekallikrein even after activation moved slower (Rf = 0.3) in electrophoresis at an alkaline pH than active kallikrein (Rf = 1). Gel filtration resolved BLM kallikrein to two proteins of low (4 X 10(4) M) and high (1.5 X 10(5) M) molecular weight. After isoelectric focusing of the activated BLM fraction, two kallikreins with pIs of 3.9 and 5.3 were obtained. The BLM fraction also contained renin which became active after Triton treatment. Renin activity was not enhanced by trypsin or acid pH indicating that there was no prorenin present. Thus, BLM of rat kidney contains a kallikrein which is different from urinary kallikrein. This kallikrein, when released from basal membrane, may appear in renal lymph and venous effluent.