By using monoclonal antibody against human pancreatic secretory trypsin inhibitor (PSTI), we developed a highly sensitive, simple, and reliable two-site enzyme immunoassay system. The minimum amount of PSTI detected by this EIA is approximately 10 pg/ml when a 100 microliter aliquot of the sample is used. Good reproducibilities of within- and between-assay series and excellent recovery of exogenous PSTI from serum were observed. The correlation between the values obtained by the EIA and RIA methods was given by the linear regression equation, y = 1.09x + 4.6, for which the correlation coefficient (r) was 0.980 (n = 20). Antigenicity of the trypsin-PSTI complexes was found to be approximately 10% of that of PSTI. From these results, it seems that our recently developed EIA system for PSTI is useful in clinical testing for quantitation of PSTI in body fluids, for biochemical studies on synthesis and secretion of PSTI, and also for study of pathophysiological mechanisms involved in the development of acute pancreatitis and certain malignant neoplasms.