Exo-beta-N-acetylmuramidase, or beta-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, is produced by Bacillus subtilis B, growing in a succinate/peptone/salts medium, at the end of exponential growth and occurs partly in the medium and partly bound to the cells. A lysozyme digest of Micrococcus lysodeikticus cell walls, O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucose and O-[2-acetamide-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose in decreasing order of efficiency, induce the enzyme but O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose does not do so. The enzyme was purified from the growth medium, after removal of the cells by continuous centrifugation, by ammonium sulphate precipitation, continuous filtration through XM-300 membranes (to remove the high-molecular-weight material which renders the enzyme sedimentable in low-ionic-strength solutions), diafiltration through PM-30 membranes and ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex. Two peaks of activity were obtained. Peak A was purified 1800-fold and was homogenous on polyacrylamide disc gel electrophoresis. A second heterogeneous fraction (peak B) was also collected. Exo-beta-N-acetylmuramidase is most stable at pH 8.0 and has a molecular weight of about 90000. The results of studies on its ability to attack several synthetic and natural substrates are given. The Km and V values for 4-methylumbelliferyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucose and O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose are respectively 0.19 and 0.65 mM and 1.50 and 16.29 mumol min(-1) mg(-1). From these results and those of inhibition studies it is concluded that the enzyme is specific for substrates with non-reducing N-acetylmuramic acid end groups. Possible roles for this enzyme are discussed.