The steroid binding properties of the human pancreatic estrogen binding protein (hEBP) in cytosol were studied by equilibrium dialysis. A high ligand specificity of the protein was revealed. hEBP in cytosol binds unconjugated steroid estrogens with a medium affinity (Kd = 10(-7) M) but does not bind conjugated estrogens or unconjugated androgens, gestagens, glucocorticoids or cholesterol. Quantitation of hEBP by radioimmunoassay in subcellular fractions of human pancreatic tissue indicated that the protein is translocated into different subcellular compartments. Duodenal juice taken from patients following stimulation of pancreatic secretion by food ingestion (Lund's test) showed high hEBP concentrations, and the levels of hEBP changed concomitantly with the levels of pancreatic isoamylase, indicating that hEBP secretion was stimulated by food ingestion. The levels of hEBP in peripheral serum from healthy subjects showed no sex difference, but were positively correlated to age. Highly elevated (10-20-fold) hEBP levels were found in serum from patients with acute pancreatitis, while normal serum hEBP values were found in other abdominal diseases. It is speculated that hEBP might have a specific role in the transport of estrogens from the peripheral circulation via the pancreas to the duodenum. The elevated hEBP levels in patients with acute pancreatitis indicate that this protein may be used as a marker of cellular damage in the pancreas.