A serine proteinase was purified from Acanthamoeba culbertsoni by 41∼80 ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE_ Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5∼7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was 37℃. It was stable for at least 16 h at 4℃ and 37℃, but it was rapidly inactivated at 65℃. The activity of the purified enzyme was not influenced significantly by Mg^(2+). Mn^(2+). Zn^(2+) or Ca^(2+). However, the enzyme activity was highly inhibited by Hg^(2+). The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.