One of several possible mechanisms for the HLA-disease association is HLA-related polymorphism of cytokine expression. However, with the exception of the tumor necrosis factors, no evidence has been found for a relationship between HLA alleles and cytokine expression. This may be because cytokine responses to commonly employed mitogens are neither antigen nor HLA dependent, and responses to recall antigens are dominated by the effect of prior antigen exposure. We reasoned that responses to alloantigens would be independent of prior antigen exposure and may therefore reveal subtle HLA- related variations in cytokine production. Here we demonstrate HLA Class II- related polymorphism of IFN-γ production in the MLR performed between 32 subjects by a novel whole-blood method. HLA DR1, 2, and 6 were associated with high, whereas DR 3, 4, 5, and 7 were associated with low IFN-Γ production. Interestingly, DQ alleles with which these DR alleles are in linkage dysequilibrium, DQ1 and DQ2 and 3, were also associated with high and low IFN-γ production, respectively. Ranking of HLA alleles according to whole-blood IFN-γ production in response to mitogen or recall antigens was similar to that in the MLR, although individual allele-related differences did not reach statistical significance. TNF-α production was significantly higher in DR 3-positive than in DR3-negative subjects, in accord with previous studies. These findings suggest that HLA Class II alleles, particularly at the DQ locus, or alternatively, genes in linkage with the, regulated IFN-γ expression by T cells. The finding of HLA allele-related polymorphism of IFN-γ production corroborates other line os evidence that regulation of IFN-γ expression contributes to HLA-associated susceptibility to immunoinflammatory diseases, in particular insulin-dependent diabetes and multiple sclerosis.