Lymphocytes from lymph nodes obtained at mastectomy in breast cancer patients have been fused with murine nonproducer myeloma cells to obtain human-mouse hybridoma cultures that synthesize human monoclonal antibodies. To date, 52 hybridoma cultures synthesizing either human IgG or human IgM have been obtained from lymph nodes of 13 patients. Ig production was stable in many of these cloned cultures through 60-200 days of observation. Levels of human Ig synthesis ranged from 0.1 to 20 microgram/ml of supernatant fluid. The immunological reactivities of the human Igs were assayed on tissue sections by using the immunoperoxidase technique. Several of the human monoclonal antibodies demonstrated preferential binding to human mammary tumor cells. One human IgM monoclonal antibody was used to discriminate between mammary carcinoma cells (from 55 of 59 patients) and normal mammary epithelial cells, stroma, or lymphocytes of the same breast. Decreased binding to some of the benign breast tumors tested and to selected non-breast adenocarcinomas was also observed. This same human monoclonal antibody, however, reacted significantly with metastatic mammary carcinoma cells in lymph nodes of breast cancer patients, with no binding to normal lymphocytes or to stroma of the same node. These studies demonstrate that stable clones of human-mouse hybridomas can be generated by using lymph nodes of mastectomy patients and that clones can be selected which synthesize human monoclonal antibodies reactive with human mammary carcinoma cells.