Bacillus polymyxa var. colistinus autolysin was purified from a 1-l culture by ammonium sulfate fractionation, gel filtration, non-binding with a DEAE-Sepharose resin, and CM-Sepharose column chromatography. SDS-polyacrylamide gel (containing B. polymyxa var. colistinus cell mall) electrophoresis of the purified autolysin gave a single band at an M-r of 23 kDa exhibiting cell wall hydrolytic activity. Identification of the specific substrate bond cleaved by the autolysin indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase. The optimal temperature for the enzyme reaction was 40-50 degrees C, and the optimal pH was 4.0, which is extraordinarily unique for amidases.