The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN-gamma. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize beta 2-microglobulin (beta 2-mu). The latter abnormality is associated with lack of beta 2-mu mRNA which remains undetectable in FO-1 cells incubated with IFN-gamma. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon of the 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene. The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system.