Monoclonal antibody HI-531 of immunoglobulin G2b subclass was produced against a human gallbladder carcinoma cell line. HI-531 was investigated for reactivity with a panel comprising ten types of different origin in fluorescence-activated cell sorter analysis. The antibody reacted with the gallbladder carcinoma cell line G-415 used for immunization and with four unrelated tumors. HI-531 was further shown, with the use of the avidin-biotin complex-immunoperoxidase technique and surgically resected tissues, to be strongly reactive with carcinoma of the gallbladder, pancreas, bile duct, and gastrointestinal tract. The antibody was reacted with several types of normal epithelial cells but often more weakly expressed than on corresponding tumors. One of six fetal lung tissues was weakly stained. All other fetal organ tissues tested showed negative staining reactions. These observations suggest that HI-531 may be of value in identifying the tumor-associated antigen expressed in gallbladder carcinoma. HI-531 immunoprecipitated the Mr 43,000 molecule from extracts of Na125I- or [35S]methionine-labeled tumor cells, but not from those of [3H]glucosamine-labeled tumor cells. In addition, cytofluorometric analysis showed that cells treated with trypsin or protease greatly decreased a reactivity to the antibody. The findings suggest that the antibody recognizes a Mr 43,000 protein molecule. Sequential immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies and analyses by nonequilibrium pH gradient and polyacrylamide gel electrophoreses showed that the Mr 43,000 molecule defined by HI-531 was not a Mr 43,000 heavy chain of HLA-A,B,C antigens detected by monoclonal antibody W6/32.