The presence of a metaphase cytogenetic abnormality (CA) is the key negative predictor of outcome in patients with multiple myeloma (MM). Gene expression profiling (GEP) of such patients showed increased expression of NY-ESO-1 compared to patients with normal cytogenetics (60% versus 31%; P = .004). NY-ESO-1 was also highly expressed in relapsing MM especially patients with CA (100% versus 60.7%; P < .001). GEP findings were confirmed at the protein level by immunostaining of marrow biopsies for NY-ESO-1. We detected spontaneous NY-ESO-1-specific antibodies by enzyme-linked immunosorbent assay in 33% of patients with NY-ESO-1+ MM, especially in CA patients (9 of 13; 70%), but in none of the NY-ESO-1- patients with MM (n = 27) or healthy donors (n = 21). Spontaneous NY-ESO-1(157-165)-specific T cells (0.2%-0.6% of CD8+ T cells) were found in the peripheral blood of NY-ESO-1+ MM with HLA-A*0201/NY-ESO-1(157-165) tetramers. These NY-ESO-1-specific T cells, when expanded, killed primary MM cells (50% lysis, effector-target [E/T] ratio, 10:1). Our data demonstrate that NY-ESO-1 is frequently expressed in MM with CA and is capable of eliciting spontaneous humoral and T-cell immunity. The pool of NY-ESO-1-specific cytotoxic T cells expands easily on NY-ESO-1 peptide stimulation and is functionally active. NY-ESO-1 should therefore be an ideal tumor target antigen for immunotherapy of patients with poor-prognosis MM.