We have established a sensitive ELISPOT assay measuring interferon γ (IFN γ) release on a single‐cell basis to detect influenza peptide‐specific CD8⁺ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA‐A1 and HLA‐A2.1 binding peptide epitopes derived from the MAGE‐1 and MAGE‐3 proteins, from the melanoma‐associated antigens tyrosinase, Melan‐A/MART‐1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA‐A2‐positive donors (75%), but only in 9 of 25 HLA‐A2‐positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma‐associated peptides were detected in 5 of 25 HLA‐A2‐positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin‐2‐ and IFNα‐containing therapy. Two of the 24 healthy donors had T cells reactive with the MART‐1 27‐35 peptide. No reactivity with the HLA‐A1‐binding peptides from MAGE‐1 or MAGE‐3 was detected in any of the HLA‐A1‐positive healthy controls or melanoma patients. These results show that the IFNγ‐ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma‐associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function. Int. J. Cancer 71: 932‐936, 1997. © 1997 Wiley‐Liss Inc.