A method for the preparation of I131-labeled anti-D eluates (I[unknown] anti-D) specific for the D antigen by adsorption and elution from intact D positive red cells is presented. A standard D antigen reference system for the quantitative comparison of the red cell D antigen from various donors was developed on the basis of kinetic studies of the effect of pH, temperature and antigen concentration on the I[unknown] anti-D reaction with the D antigen. The red cell D antigen determinations of the blood from 33 Negro and 102 Caucasian donors showed wide monomodal frequency distributions which indicate a heterogeneity of quantitative D antigen reactivity consistent with previous serologic and I131-labeled anti-D studies. The average red cell D antigen reactivity of the 20 probable D homozygote and 14 established D hemizygote Caucasian donors was similar to the D antigen reactivity of the 102 random Caucasian donors. The red cell D antigen reactivity was strongly depressed by the C antigen (or C gene) in Caucasian bloods and enhanced by the E antigen (or E gene) in Negro bloods. The E antigen (or E gene) may also have enhanced the D antigen reactivity in red cells from Caucasian donors, but the difference in D zygosity provides an alternative explanation for the results. The marked influence on red cell D antigen reactivity in the presence of the C and E antigen may well have obscured any lesser influence of D zygosity.