Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cell-surface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers.