In addition to Ag presentation for T cell surveillance, MHC molecules have been implicated in mediating regulatory signals. We have assessed biologic responses following engagement of the TCR accessible region of the HLA class I alpha 2 domain. mAbs directed to this domain specifically induced cell aggregation of normal hematopoietic and leukemic cells. The functional consequences were unique since other mAbs reactive with HLA class I residues outside the TCR binding domain did not induce cell aggregation. The adhesion response required ATP, mRNA, protein, and actin synthesis and did not depend on LFA-1/ICAM interactions. Cell aggregation was also induced when all but four of the intracytoplasmic residues of the class I molecule were deleted, indicating that transduction of signals leading to cell adhesion does not require this portion of the molecule. mAbs directed to HLA class I alpha 2 amino acid residues within the TCR binding domain were also able to inhibit proliferation of normal mitogen-stimulated T cells. Growth inhibition correlated with down-regulated expression of CD25, CD28, and CD95, suggesting that reduced transduction of costimulatory signals is involved. Although HLA class I signals inducing cell aggregation required engagement of positions within the TCR binding region, growth inhibitory signals could be generated through positions both within and adjacent to this domain. Taken together, engagement of specific positions within the TCR binding domain of the class I alpha 2 helix results in active cellular responses. Thus, this region may be directly involved in signal transduction following CTL recognition of target cells.