The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13- acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.