Homozygous typing cell defining HLA-Dw2 and that defining HLA-Dw12 are mutually strongly MLC stimulatory, although both of them are typed as HLA-DR2 by conventional serology. In view of the recent findings that there exist multiple HLA-D region encoded molecules, it is possible that the mutual MLC stimulation between them are caused by molecules other than HLA-DR2. To test this possibility, two-dimensional (2-D) gel analyses using monoclonal antibodies (MoAbs) were performed for cell lines homozygous for HLA-Dw2 and HLA-Dw12. When the MoAb HU-30, monospecific for HLA-DR2, was used for immunoprecipitation, both cell lines showed identical 2-D patterns consisting of a set of heavy and light chains corresponding to HLA-DR2 antigen. In contrast, HU-20, which primarily recognized HLA-DR antigens, but was crossreactive to Ia-like antigens other than HLA-DR, exhibited more complex 2-D patterns being comprised of several sets of heavy and light chains, and the two cell lines were clearly different in their light chains. These differences in the light chains were further confirmed by using HU-11 detecting Ia-like molecules other than HLA-DR. Thus, HLA-Dw2 and HLA-Dw12 differ in Ia-like molecules other than HLA-DR2, when assessed by 2-D gel analyses. Several autoimmune diseases, in which immune response (Ir) genes are supposed to play a primary role in pathogenesis, are known to correlate significantly with the presence of certain HLA-D specificities. For example, Takayasu disease is associated with HLA-Dw12, while multiple sclerosis with HLA-Dw2. Since Ia-like antigens represent Ir gene products, the present study gives a molecular basis for different disease associations observed for HLA-Dw2 and HLA-Dw12.