Current evidence suggests that recognition of Ag/MHC by the TCR alone is insufficient to lead to T cell proliferation or effector function. For a Th cell to produce sufficient IL-2 to allow autocrine-driven clonal expansion, there is a requirement for so-called "costimulatory" or "accessory" signals in addition to TCR ligation by Ag/MHC. Although the first signal delivered by TCR ligation has been well characterized, information regarding the biochemical nature of second signals is limited. In the present report, using a newly generated mAb specific for CD28, signal transduction by the CD28 receptor has been studied in mouse splenic T cells. When freshly isolated splenic T cells were assessed, cross-linking of CD28 by mAb did not induce increases in intracellular calcium concentration were assessed, cross-linking of CD28 by mAb did not induce increases in intracellular calcium concentration whereas TCR cross-linking was able to induce calcium mobilization. In contrast, when T cells were activated by phorbol ester treatment or by in vitro culture, CD28 ligation was able to induce calcium mobilization in 60 to 70% of splenic T cells. Unexpectedly, the CD28-induced calcium response was mainly limited to T cells of the CD4+ subset, whereas both CD4+ and CD8+ T cell subsets showed increases in [Ca2+]i of similar magnitude after CD3 epsilon ligation. The temporal nature of the CD28-induced signal was also different from TCR-induced calcium mobilization. CD28-induced signals were delayed in onset and sustained in duration in contrast to TCR signals that had short latency and brief duration. Differential expression of CD28 on the surface of activated CD4+ or CD8+ T cells did not appear to account for the differences in signal transduction between the two T cell subsets. The preferential responsiveness of the CD4+ T cell population to CD28-induced signaling was also observed in downstream events such as the induction of IL-2R, CD69 expression, and in cellular proliferation. These results indicate that the costimulatory signal delivered by CD28 may have fundamentally different biochemical properties in CD4 and CD8 T cell subsets, and therefore the functional role of CD28 may differ in these T cell subsets.