NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD8(+) T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD8(+) T cell responses to NY-ESO-1, (ii) to determine the frequency of CD8(+) T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD8(+) T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD8(+) T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines.