Histocompatibility leukocyte antigen (HLA)-A2 is used as a restricting element to present several melanoma-associated antigen (MAA)-derived peptides to cytotoxic T lymphocytes (CTLs). HLA-A2 antigen is selectively lost in primary melanoma lesions and more frequently in metastases. Only scanty information is available about the molecular mechanisms underlying this abnormality, in spite of its potentially negative impact on the clinical course of the disease and on the outcome of T cell–based immunotherapy. Therefore, in this study we have shown that the selective HLA-A2 antigen loss in melanoma cells 624MEL28 is caused by a splicing defect of HLA-A2 pre-mRNA because of a base substitution at the 5′ splice donor site of intron 2 of the HLA-A2 gene. As a result, HLA-A2 transcripts are spliced to two aberrant forms, one with exon 2 skipping and the other with intron 2 retention. The latter is not translated because of an early premature stop codon in the retained intron. In contrast, the transcript with exon 2 skipping is translated to a truncated HLA-A2 heavy chain without the α₁ domain. Such a polypeptide is synthesized in vitro but is not detectable in cells, probably because of the low steady state level of the corresponding mRNA and the low translation efficiency. These results indicate that a single mutational event in an HLA class I gene is sufficient for loss of the corresponding allele. This may account, at least in part, for the high frequency of selective HLA class I allele loss in melanoma cells. Our conclusion emphasizes the need to implement active specific immunotherapy with a combination of peptides presented by various HLA class I alleles. This strategy may counteract the ability of melanoma cells with selective HLA class I allele loss to escape from immune recognition.