The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.