OBJECTIVE To develop and validate a radioimmunoassay for feline trypsin-like immunoreactivity (fTLI). METHODS A double antibody radioimmunoassay for determination of fTLI was developed, using feline trypsin purified from feline pancreatic tissue antiserum against feline trypsin raised in New Zealand White rabbits, and a commercially available precipitation solution. This assay was validated by determining detection limit, linearity, accuracy, and intra- and interassay variabilities. A control range was determined from samples of 21 clinically normal cats. RESULTS The detection limit for fTLI in this assay was 1.9 micrograms/L. The linearity of the assay was evaluated by testing dilutional parallelism. Observed/expected values ranged between 95 and 127%. The accuracy of the assay was tested by measuring recovery of added trypsin. Observed/expected values ranged from 99 to 114%. Intra-assay variability ranged between 3.0 and 9.4%, and interassay variability ranged between 4.0 and 8.2%. The control range for serum fTLI was determined to be 17.1 to 48.6 micrograms/L. CONCLUSIONS The assay for fTLI described here is sensitive, linear, accurate, precise, and reproducible. CONCLUSIONS This assay may be of use for the diagnosis of exocrine pancreatic diseases in cats. Preliminary results of such research have been promising, and further clinical studies are in progress.