In this report we report the identification of novel molecular associations involving the MHC class II (Ia) Ag expressed on the surface of Ag-presenting B lymphocytes. Biosynthetically radiolabeled murine B cells were incubated for 2 h in the presence or absence of T lymphocytes before treatment with the cleavable cross-linking reagent dimethyl-3,3'-dithiobispropionimidate. Anti-Ia immunoprecipitates of solubilized cell extracts revealed novel cross-linked products of Mr 90,000 to 95,000 which, upon cleavage of the cross-linker, could in part be resolved into native Ia and other structures of approximately Mr 67,000. The detection of the cross-linked products was significantly enhanced in B cells that had been co-cultured with T lymphocytes, but not with other cell types, and co-culture with various monoclonal T cell lines resulted in different levels of enhancement. Detection of the 90- to 95-kDa cross-linked products appeared to be independent of the foreign Ag for which the T cells were specific and could be enhanced when either cell type was replaced by a plasma membrane fraction, indicating that it resulted from direct cell-cell contact. These results suggest that some proportion of the Ia glycoproteins expressed on the surface of B cells become associated with other structures of Mr 67,000 upon Ag-non-specific interactions between T and B lymphocytes.