B cells derived from peripheral‐blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from a patient with a high serum antibody titer to autologous melanoma were transformed with Epstein‐Barr virus (EBV) and evaluated for reactivity against autologous tumor. B cells producing antibody reactive with autologous tumor and unreactive with normal fibroblasts were detected both in TIL and in PBL. One cell line derived from PBL and another derived from TIL sustained production of tumor‐reactive antibody for 10 weeks and over 15 months respectively. The cell line derived from PBL, 2D11, produced an antibody reactive with a trypsin‐resistant antigen expressed on the cell membrane of autologous and allogeneic melanoma cell lines. The cell line derived from TIL, IF6, produced an antibody reactive with a cell‐surface glycoprotein expressed by 5 autologous melanoma cell lines derived from 5 different metastases and 16/19 allogeneic melanoma cell lines. IF6 also showed reactivity with cell lines derived from a blue nevus, a congenital nevus, an astrocytoma, and 1/4 renal‐cell carcinomas; but it was not reactive with 5 foreskin melanocyte cell lines, 2 normal fibroblast lines, 5 leukemia/lymphoma lines, 8 lung‐cancer lines, 8 glioblastoma lines, or lines derived from 1 ovarian carcinoma, f colon carcinoma, 1 vulvar carcinoma, 1 fibrosarcoma, 1 murine melanoma, or 4 murine leukemia/lymphomas. We describe here an antibody that detects a new melanoma specificity obtained by EBV transformation of tumor‐infiltrating B cells. © 1992 Wiley‐Liss, Inc.