Saccharomycopsis lipolytica CX161-1B, a strain suitable for genetic studies, when grown at neutral pH produced a single alkaline extracellular protease, lower levels of acid extracellular protease(s) and no neutral extracellular protease. The alkaline protease was purified to homogeneity (as determined by polyacrylamide gel electrophoresis) by ultrafiltration, gel filtration and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated by gel filtration to be 27000-30000, and the isoelectric point was pH 5.7. The purified enzyme had an alkaline pH optimum (pH 9-10). It was completely inhibited by phenylmethylsulphonyl fluoride, reversibly inhibited by EDTA, partially inhibited by o-phenanthroline, and not inhibited by dithiothreitol, N-ethylmaleimide or 4-hydroxymercuribenzoic acid, indicating that it is a serine protease. The content of sulphur amino acids was determined, and the purified protease contained no more than 1.8% carbohydrate as determined by the phenol-sulphuric acid method. The N-terminal amino acid sequence (25 residues) was determined; the N-terminal amino acid was alanine.