Direct antiglobulin (Coombs') tests of erythrocyte (RBC) subpopulations confirmed the presence of membrane-bound immunoglobulin G (IgG) on old (density > 1.110) human RBCs but not on the young (density > 1.110) RBCs. After thermal elution of the bound IgG, this Coombs' reaction was negative, but incubation of thermally eluted IgG (He-IgG) with heat-treated RBCs indued a positive antiglobulin reaction was also obtained after incubation of heat-treated RBCs with anti-T antibody. Similar results were obtained when young RBCs treated with Vibrio cholerae neuraminidase (VCN) were incubated with anti-T or with IgG eluted by heat from old RBCs. Nevertheless, pre-absorption of heat-eluted IgG with T and/or Tn antigen, did not prevent it from binding to either heat-treated old or VCN-treated young RBCs as assessed by the antiglobulin consumption assay. Pre-treatment of either VCN-treated young or heat-treated old RBCs with anti-T and/or anti-Tn antibodies had no significant effect on the binding of radiolabeled He-IgG (eluted from old RBCs). The results indicate that even though desialylation of the erythrocyte membrane is required for binding of both anti-T/-Tn and He-IgG, the specificity and consequently the RBC binding sites for He-IgG and anti-T seem to be different.