The human major histocompatibility complex comprising the HLA class I and II genes provides a versatile source of natural heterozygous loci. This polymorphic genetic system allows analysis of the mechanistic aspects of loss of heterozygosity (LOH), a major phenomenon observed at tumor suppressor genes in human cancer cells. Four lymphoblastoid cell lines, ORI, TK6, WI-L2-NS and VH, were used to adjust current HLA immunoselection protocols to quantify loss of HLA-A2 in human lymphoblastoid cell lines. The modified selection protocol was used to isolate independent spontaneous HLA-A2 mutants from the lymphoblastoid cell line ORI. The frequency of spontaneous loss of HLA-A2 in ORI was 1.7 x 10(-5). By HLA typing 35 spontaneous HLA-A2 mutants, we showed that 74% of the HLA-A2 mutants also lost expression of the HLA-B allele, which is located on the same haplotype as HLA-A2. Microsatellites on both arms of chromosome 6 were used for molecular characterization of the spontaneous HLA-A2 mutants. Loss of heterozygosity at various loci on the p-arm or loss of an entire chromosome 6 was found in 80% of the mutants. Surprisingly, it appeared that a presumed mitotic recombination event in the cell line ORI itself had resulted in homozygosity of all markers distal from the HLA locus up to the telomere. This greatly limited the detection of mitotic recombination, resulting in LOH up to the telomere, on the short arm of chromosome 6 in this cell line. However, gene dosage analysis detected two copies of the remaining D6S265 allele in mutants which showed LOH at various loci along the p-arm. This suggested that recombination resulted in LOH in these mutants. The lymphoblastoid cell line TK6 did contain informative microsatellites along the complete chromosome 6. Mutants of TK6 either retained heterozygosity of all p-arm markers, showed LOH of all p-arm markers or showed loss from a breakpoint up to the telomere. These data indicate that recombination and chromosome loss both are important mechanisms involved in loss of the HLA-A2 allele in vitro. Such mechanisms may be involved in LOH in vivo and contribute to loss of tumor suppressor alleles.