The monoclonal antibodies 109d6 and IVD12 reacted with separate polymorphic Ia epitopes in immunofluorescent studies. Using a panel of lymphoblastoid B cell lines, antibody 109d6 reacted with all HLA-DR4 or DR7 positive lines in a pattern resembling the MT3 specificity recognized by human alloantisera. The antibody IVD12 reacted with all HLA-DR4 and two of three DR5 positive B cell lines suggesting that it recognized a specificity similar to MB3. The intensity of fluorescence was greater on DR5(+) cell lines than on DR4(+) cell lines relative to the amount of a nonpolymorphic Ia determinant. Among 45 unrelated control individuals reactivity with antibody 109d6 was correlated most closely with (r = 0.724) but not identical in occurrence to the MT3 specificity. Cocapping experiments demonstrated that the 109d6 epitope and the IVD12 epitope were present on independently redistributed cell surface molecules of DR4 homozygous lymphoblastoid cell lines. Furthermore, the DR4 alloantigens detected by an absorbed polyclonal human alloserum were similarly identified on molecules that were independent from those bearing either the 109d6 epitope or the IVD12 epitope. Taken together, these data indicate the existence of at least four distinct, serologically defined Ia molecular species.