The extract from cotyledons of 6-day-old peanut seedlings contained an enzyme that decomposed the chromogenic substrate N-benzoyl-D,L-arginine-p-nitroanilide (BAPA). The enzyme was purified by ammonium sulfate precipitation, pH adjustment, and anion-exchange chromatography. The molecular weight was found to be 60 000 as estimated from the Stokes radius obtained by gel filtration on Sephadex G 200. Its temperature response curve showed optimal activity between 25 and 36° and its pH optimum was 7.4. Anion-exchange chromatography as well as polyacrylamide gel electrophoresis of the purified extract resulted in two distinct areas of activity with regard to BAPA hydrolysis. Inhibitor experiments revealed that the enzyme does not have sulfhydryl groups at its active site nor does it respond to specific trypsin inhibitors, but it was inactivated by diisopropylfluorophosphate. It is therefore likely that it is a serine-type peptidase.