Two Monoclonal Antibodies produced against Pancreatic Amylase have been produced and used to develop immunoassays (MAPA assays) for serum isoamylases. One antibody binds pancreatic isoamylase specifically and neither antibody inactivates amylolytic activity. MAPA assays that measure pancreatic and total enzymatic activities directly and rapidly in hyperamylasemic serum are described in this report. The influence of isoamylase determination on diagnosis is also investigated in 35 hyperamylasemic patients. The percentage for pancreatic amylase in serum as determined by immunoassay correlates well with results obtained by conventional electrophoresis (r = 0.90). Chart review indicates that use of MAPA assays would have potentially changed the diagnosis in 27% of patients. Five of six additional patients with hyperamylasemia caused by macroamylasemia had normal immunoreactive amylase values, and in all six patients the values were strikingly lower than predicted. The ability to assay organ-specific pancreatic isoamylase directly and early in the clinical course should reduce the risks of misdiagnosis and inappropriate therapy, especially in salivary-type hyperamylasemia or macroamylasemia.