Historically, serum amylase and lipase levels have been used to indicate pancreas injury; however, these enzyme levels are often not predictive of pathology. In an effort to discover novel biomarkers of pancreatic acinar cell injury, we analyzed serum and pancreas tissue from cyanohydroxybutene (CHB)-treated male IGS rats using proteomics methods. CHB produces an "edematous pancreatitis," characterized by depletion of zymogen granules, acinar cell apoptosis, and mild to moderate inflammation. Secondary necrosis occurs at higher doses. Rats were treated with 150 mg/kg of CHB and samples were collected at 4, 8, and 24 h. Analyses of serum tryptic digests by surface-enhanced laser desorption-ionization mass spectrometry revealed two novel peptide biomarkers (RA1609 and RT2864) that were predictive of pancreatic damage. Levels of RA1609 decreased, while levels of RT2864 increased by 8 h following CHB treatment. The changes in RA1609 and RT2864 were detected in media from CHB-treated primary rat acini, demonstrating that these peptides are either of pancreatic cell origin or are produced by proteases released from acinar cells. Sequencing revealed that RA1609 is a fragment of rat albumin (accession number P02770, residues 348-360) and RT2864 is a portion of either rat trypsin III (accession number P08426, residues 39-65) or bovine trypsin (accession number P00760, residues 35-61). These two peptides, and possibly other fragments of serum proteins that are digested by pancreatic proteases, may be useful as safety markers for exocrine pancreatic toxicity during drug development or as biomarkers for the diagnosis and/or grading of severity of pancreatic disease.