Many types of human and animal tumors have an absolute requirement for methionine. This requirement can be satisfied by homocysteine only in normal cells and tissues. Therefore, methionine may be an important target in cancer therapy. To attack this target we have purified endotoxin-free methioninase from Pseudomonas putida by a novel and simple procedure. This procedure involves (1) a heat step of the cell extract at 60 degrees C for 8 min, (2) DEAE-Toyopearl ion-exchange chromatography, (3) DEAE-Sephadex A50 ion-exchange gel filtration chromatography, and (4) affinity chromatography on Acticlean to remove the endotoxin bound to the enzyme. The yield for this purification was up to 80%. The methioninase has four subunits of approximate molecular weight 43 kDa. This is the first methodology for methioninase that allows rapid purification with high yield and separation from endotoxin suitable for in vivo efficacy testing against methionine-dependent tumors in animal models.