Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.001). Increased lipase levels were noted in 10% of patients and in only 3% of siblings and 2% of control subjects (p < 0.001). Using both univariate and multivariate statistical analysis, elevated lipase activities at clinical onset were associated with higher titers of autoantibodies against islet cell cytoplasmic antigens and glucagon, but not against insulin or the 65-kDa isoform of glutamic acid decarboxylase (GAD65-Ab), or with markers of genetic predisposition or metabolic dysregulation. These findings indicate the presence of modest, but statistically significant, variations in circulating pancreatic enzyme levels in 28% of IDDM patients at clinical onset (p < 0.001 vs. 5% in control subjects). Increased lipase levels may express a form or a stage of the disease with exocrine cell damage; their association with higher titers of islet cell and glucagon autoantibodies is not yet explained. Lower lipase and isoamylase levels are thought to result from the reduced acinar cell function in the vicinity of insulin-depleted islets. It must be tested whether pancreatic enzyme activities in serum can also be altered during the preclinical stage and can thus be considered as an additional marker for the disease process in the pancreas.