This study reports the identification of a new gene, designated Melan-A, that encodes an antigen recognized by autologous cytolytic T lymphocytes (CTLs) on HLA-A2 melanomas. The antigen, named LB39-Aa, was initially identified in a CTL clone isolated from blood lymphocytes of a HLA-A2 melanoma patient (LB39). The gene, which is unrelated to any known gene, is 18 kb long and comprises five exons. It is expressed in most melanoma tumor samples and only in melanocytes among normal cells. The identification of Melan-A was achieved through a cDNA library transfection approach in COS cells, which is faster and more convenient than the cosmid transfection method used for identifying other tumor antigens. The Melan-A gene is the third gene found to code for an antigen recognized by autologous CTLs on human tumors, following the tyrosinase gene and MAGE-1. These findings provide a basis for understanding the recognition of melanocytic differentiation antigens by antitumor CTLs in melanoma patients.This study reports the identification of a new gene, designated Melan-A, that encodes an antigen recognized by autologous cytolytic T lymphocytes (CTLs) on HLA-A2 melanomas. The antigen, named LB39-Aa, was initially identified in a CTL clone isolated from blood lymphocytes of a HLA-A2 melanoma patient (LB39). The gene, which is unrelated to any known gene, is 18 kb long and comprises five exons. It is expressed in most melanoma tumor samples and only in melanocytes among normal cells. The identification of Melan-A was achieved through a cDNA library transfection approach in COS cells, which is faster and more convenient than the cosmid transfection method used for identifying other tumor antigens. The Melan-A gene is the third gene found to code for an antigen recognized by autologous CTLs on human tumors, following the tyrosinase gene and MAGE-1. These findings provide a basis for understanding the recognition of melanocytic differentiation antigens by antitumor CTLs in melanoma patients.