Absorption experiments and subsequent retesting on human peripheral lymphocytes were performed with the congenic anti-H2f mouse serum known to give strong cytotoxic reactions with human cells which correlate with the presence of HLA-A2 antigen (mouse serum ASP 223, donor strain B10.M (H-2f), recipients (B10 X A.SW)F1 (H-2b/H-2s) hybrids; antibodies present in the serum anti-H-2.9, 8,37). Depending on the dose of A2-negative cells used for absorption, the serum can be rendered operationally monospecific to HLA-A2. Absorption experiments with lymph node cells of different mouse strains have shown that H-2f.p.w7 haplotypes share the ability to absorb completely the antihuman activity with about 15 X 10(6) cells. Consequently, the antihuman activity is not attributable to the presence of the anti-H-2.9 private specificity in the serum. H-2d.k.r haplotypes have weak absorbing capacity, because 500 X 10(6) lymph node cells are needed to absorb 70% of the antihuman cytotoxic acitivity. All strains that were able to absorb antihuman cytotoxic activity from ASP 223 shared the specificity H-2.8. Thus, the H-2.37 specificity seems to be responsible for strong and the H-2.8 specificity for weak absorbing capacity. The absorbing capacity of the H-2d haplotype was localized in the H-2.K end. Mouse lymph node cells have a 10- to 20-fold higher absorbing capacity for the antihuman activity than thymus cells. It was shown that H-2.K end public specificities predominantly expressed on lymph node cells are responsible for the generation of antihuman (anti-HLA) cytotoxic activity in anti-H-2 sera.