The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced (1,2) and anti-CD2-induced (3) activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig kappa "second step" monoclonal antibodies) increased the capacity of anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig kappa) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.