Unreliable results were obtained in an enzymatic assay of total calcium (Ca) in a patient's serum using porcine pancreatic amylase (PPA). Besides, the result was different from that obtained by the atomic absorption analysis. Fractionation of the patient's serum by ultrafiltration and Sephadex G-200 gel filtration revealed the presence of an interference substance with a large molecular size of more than 200kDa. This interference substance was absorbed on a Protein A column. Furthermore, an immune precipitate was observed between the patient's serum and PPA by Ouchterlony double immunodiffusion. An antibody against PPA may exist in the patient's serum and interfere with the enzymatic assay of Ca using PPA.