Recently, a radioimmunoassay (RIA) for measurement of canine pancreatic lipase immunoreactivity (cPLI) in serum was developed and validated. However, RIAs require frequent use of radioactive materials. Therefore, the goal of this project was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for cPLI. After purifying cPL, we developed and purified antiserum against cPL in rabbits. The purified antibody was bound to microtitre plates and used to capture antigen. A portion of the purified antibody was biotinylated and used to identify the captured antigen. Streptavidin labelled with horseradish peroxidase and a horseradish peroxidase substrate were used for detection. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. The reference interval for serum cPLI was determined by the central 95th percentile in 74 clinically healthy dogs: 2.2 to 102.1 microg/L. The sensitivity and the upper limit of the working range were 0.1 and 999.2 microg/L, respectively. The ratios of observed to expected values for dilutional parallelism for 6 serum samples ranged from 0.0 to 148.8%; the ratios for spiking recovery for 4 serum samples ranged from 90.4 to 112.6%, assuming 55% recovery of the cPL. Coefficients of variation for intra- and interassay variability for 6 different serum samples were 2.4, 3.4, 4.1, 5.8, 7.4, and 10.0% and 5.9, 7.7, 11.6, 13.9, 23.5, and 46.2%, respectively. We conclude that the ELISA described here is sufficiently sensitive, linear, accurate, precise, and reproducible for clinical application. Evaluation of its clinical usefulness for the diagnosis of exocrine pancreatic disorders in dogs is under way.