We performed molecular analysis of complement components (C3, C4, and factor B) in human bile by sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulfide bonds, and C3 levels ranged from 45 to 650 micrograms/ml (n = 15). C4 was detected as a triple chain (98-kDa alpha-chain, 73-kDa beta-chain, and 33-kDa gamma-chain) molecule linked by disulfide bonds, and C4 levels ranged from 2.5 to 60 micrograms/ml. Factor B, a component of the alternative pathway, was also detected, as an intact form. Factor B levels ranged from 0.3 to 8.0 micrograms/ml. The sizes and subunit structures of complement components in human bile were compatible with those reported in human serum. The results of a hemolytic assay indicated that complement molecules in human bile were functionally active. These molecules may participate in local immune and inflammatory responses in the biliary tract.