(1) Human trypsin was purified from human pancreatic tissue and used as an immunogen in the production of monoclonal antibodies. It was also used as a standard preparation of human trypsin in the screening of monoclonal antibodies and in the development of an immunoradiometric assay (IRMA). (2) Eight monoclonal antibodies were raised to human trypsin, two of which were subsequently employed in the development of an IRMA to measure immunoreactive trypsin (IRT) in blood and blood spots. (3) An antiserum to human trypsin was raised in sheep and employed in a sandwich ELISA. (4) Monoclonal antibodies were tested by a number of different screening systems including ELISA, immunoblotting and immunoadsorption assays with 125I-labelled human trypsin. (5) Six selected monoclonal antibodies were tested for use (a) as the solid phase antibody and (b) as the radioactive tracer in an IRMA, and against each other for compatability in an IRMA. (6) One combination of antibodies was chosen and experiments performed to determine (a) lack of competition of binding to antigen between the two and (b) positive binding of the combination to serum IRT. (7) An IRMA to measure IRT in blood and blood spots was developed using two monoclonal antibodies, 56/C5/33 (IgG2a) and 125I-labelled 55/A4/31 (IgM). (8) The detection limit of the assay with purified cationic human trypsin as the analyte was determined to be 0.8 ng trypsin. (9) The IRMA was performed with reconstituted blood containing standards of human trypsin (either blood or dried blood spots on filter paper). (10) There is potential for this assay to be further developed and employed as a routine screening assay to detect elevated concentrations of IRT in dried blood spots from newborn infants with cystic fibrosis.