The expression of HLA class I molecules on tumor cells is vital for CD8+T cell recognition of tumor Ags. Loss of HLA class I Ag expression as a result of defective beta 2-microglobulin genes has been described in melanoma cells. To further evaluate mechanisms of tumor escape, HLA class I Ag expression was compared in 24 metastatic melanoma cell lines and 20 melanocyte strains by FACS analysis with use of allele-specific mAbs. Total loss of HLA class I Ag expression was not noted; instead, two relatively common phenomena were identified: 1) A variable degree of expression of HLA-B Ags by melanoma cell lines and melanocytes; however, HLA-A Ags were consistently expressed in all cell types. Furthermore, HLA-B locus Ag expression was detected in vivo in only one of six frozen section specimens obtained from six patients having metastatic melanoma. 2) Loss of allelic expression was noted in two of 14 HLA-A2 (14%) and one of three HLA-A29 (33%) melanoma cell lines and included a full haplotype, which suggests loss of a genomic fragment. Allele-specific PCR amplification demonstrated deletion of genes in linkage disequilibrium within the MHC class II, III, and I regions. Aberrations of HLA class I expression in tumor lines should be considered when assessing MHC-restricted phenomena in in vitro models.