An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45 degrees C; activity declined rapidly at higher temperatures but significant activity persisted down to 4 degrees C. Activity was strongly inhibited by 10(-3) M EDTA and was partially restored by addition of Zn2+, Ca2+ or Co2+. The Km values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a mild salts buffer at 55 degrees C but was very stable in the latter at more than 80 degrees C.