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Lactoferrin (LF), chymotrypsin and lipase activity were measured in duodenal juice during pancreatic stimulation. Secretin (0.5 CU/kg/h) plus cerulein (75 ng/kg/h) were infused intravenously in 98 subjects: 33 patients without organic diseases (C), 40 patients affected by chronic pancreatitis (CP), and 25 patients with different gastrointestinal diseases (GID). LF was determined by means of a new noncompetitive immunoenzymatic assay with a sensitivity in the duodenal juice of 5 ng/ml. Duodenal LF concentrations were significantly higher in CP than in C or GID (p < 0.001). LF was in a normal range in acute relapsing pancreatitis due to biliary stones or pancreas divisum. In the diagnosis of the chronic pancreatitis, LF/lipase ratios showed a specificity of 93% and a sensitivity of 95%. Our results show that LF immunoassay in duodenal juice is a sensitive and accurate assay to apply in pancreatic function tests involving duodenal content analysis.