The recent solutions of the MHC class II crystal structure reveal dimerization of the alphabeta heterodimers. These dimer of dimers structures may also exist either on resting cells or after engagement by TCR, and may be involved in B cell signaling and up-regulation of co-stimulatory molecules such as B7 which facilitate T cell activation. By combining crystallographic data on HLA-DR1 with the sequence of murine I-Ak and refining the resulting structure through energy minimization calculations, we have predicted the contact amino acids expected to stabilize the I-Ak dimer of dimers structure. As in HLA-DR1, three salt bridges in I-Ak (D alpha62-Hbeta112, H alpha181-E beta163, E alpha183-Hbeta113) appear to provide the main interaction. Guided by this structural data, we prepared 45 B cell transfectants representing 20 different class II mutation phenotypes in the contact region containing these salt bridges. We examined their abilities to activate three T cell hybrids. Antigen-specific h4Ly50.5 cells were not greatly affected by changes in the dimer of dimer contact residues. In contrast, autoreactive C8.A3 T cells were very sensitive to changes in this region but presentation of class II of many mutation phenotypes could be rescued by treatments that up-regulate B7-1. The alloreactive hybridoma 2H40.2.5 was less sensitive to changes in the contact residues. A simple model was developed that summarizes the effects of the mutations for the T cells tested. Mutations at D alpha162, E alpha183, H alpha181 and Rbeta106 had the largest negative impact, while D alpha166, E alpha185, Hbeta112, Hbeta113 and E beta163 were less disruptive. Results are consistent with mutations interfering with class II interaction with another molecule which might or might not be another class II heterodimer. However, the larger negative impact of alpha chain mutations in salt bridge pairs suggests that these sites also help maintain some essential conformation of the alpha chain apart from any possible impact on dimer of dimers stability.