A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34-41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%-16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life=7.3 h) than that from SBL11 (half-life=6.4 h at 50 degrees C), while both showed the same temperature optimum of 37 degrees C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.