The presence of immunoreactive atrial natriuretic peptide (irANP) in rabbit, pig and human gallbladders was investigated using radioimmunoassay and immunohistochemistry. Serial dilution curves of gallbladder tissue and bile juice extracts were paralleled to the standard curve of atriopeptin III. Gel filtration profiles of gallbladder tissue extracts showed a major peak corresponding to rat pro-ANP. The amounts of irANP in rabbit, pig and human gallbladders were 30.0 +/- 12.3 pg/mg (n = 7), 7.0 +/- 2.0 fg/mg (n = 7) and 17.7 +/- 2.0 fg/mg wet tissue (n = 8), respectively. Bile juice was also shown to contain irANP but with small molecular mass. The amounts of irANP in the rabbit, pig and human bile juice were 25.0 +/- 2.0 (n = 7) and 0.50 +/- 0.02 (n = 7), and 1.3 +/- 0.1 pg/ml (n = 8), respectively. The immunohistochemical staining of the rabbit gallbladder tissue revealed the presence of irANP in the luminal epithelium and smooth muscle layer. The amount of irANP was higher in the luminal epithelium than in the rest of the gallbladder tissue from rabbits (0.30 +/- 0.06 vs. 0.01 +/- 0.01 pg/microgram protein, P < 0.01). These findings suggest that ANP may be synthesized and stored in the gallbladder, and may have a role in the regulation of fluid balance and cystic motility.