Primary breast cancers from 19 patients and draining lymph nodes from nine of them (seven containing metastatic tumor) were used in growing tumor-infiltrating lymphocytes (TIL) in culture. TIL were studied for proliferation, phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor. Lymphocytes from primary breast tumors proliferated in 15 of 19 cultures, a median of 6.7 x 10(3)-fold in 65 days. For eight of nine patients, lymphocytes derived from draining lymph nodes proliferated in culture, a median of 110-fold in 49 days. Breast TIL became predominantly CD4+ cells over time in culture and were 73% CD4+ and 21% CD8+ (means) at 63 days (median). Lymph node lymphocytes were 63% CD4+ at 51 days. TIL were poorly lytic in 4-hour 51Cr release assays. Lysis of autologous tumor occurred in only one of 12 breast TIL and one of nine lymph node cultures. This lysis was low (15% at effector:target = 40:1) and was nonspecific (non-major-histocompatibility-complex restricted). Cytokine secretion was tested by co-culturing TIL with autologous or allogeneic tumors for 24 hours. Cytokines were measured in culture supernatants by enzyme-linked immunosorbent assay or radioimmunoassay. TIL from three of 11 patients specifically secreted granulocyte macrophage-colony-stimulating factor, tumor necrosis factor-alpha and interferon-gamma when stimulated by autologous tumor and not by a panel of four to five allogeneic breast cancers. Cytokine secretion has made possible the identification of lymphocytes infiltrating breast cancers with specific immune reactivity. This finding will guide the development of new immunotherapies for patients with breast cancer.