The cross-linking of the cell surface molecule by its specific antibody mimics the binding of the ligand to the receptor and leads to the aggregation and clustering of molecules. This study is undertaken to investigate the signal-transducing potential of CD27 and the engagement of CD27 by cross-linking with anti-CD27 antibody. We investigated 1) whether CD27 cross-linking along with CD3 cross-linking would differentially affect different T cell subsets, and 2) whether signals delivered via CD3 plus CD27 and CD28 plus CD27 in CD45RA and CD45RO T cells could act synergistically on the activation of transcription factors, cytokine synthesis, proliferation and overall induction of T cell activation. We analyzed the intracellular signaling events and the data showed that co-cross-linking with both co-receptors induces p38 and IL-4 secretion in CD4+CD45RO+ cells, and ERK2 and IFN-γ in CD4+CD45RA+ cells in an antigen-independent manner. Various subsets of T cells were isolated from PBMC by negative selection using Dyanl magnetic beads. Purified cells were stimulated through receptors by cross-linking with antibodies. MAP kinases, STAT6, STAT1, GATA3 and T-bet were analyzed by western blotting. Cytokine profiles by ELISA and cell proliferation by MTT Assay were analyzed. CD3XL+CD27XL and CD28XL+CD27XL induced Th2 phenotype in CD4+CD45RO+ cells and similar cross-linking resulted in Th1 phenotype in CD4+CD45RA+ cells. It is also clear from these studies that p38MAPK, STAT6, and GATA3 appear to be associated with Th2 phenotype, and MAP kinase ERK2, STAT1, T-bet and IFN-γ appear to be associated with Th1 phenotype. We also observed a positive relationship between activation of p38MAPk and IL-4 synthesis by utilizing p38MAP kinase inhibitor SB203580. Our data suggests that CD4+CD45RA+ cells act as TH1 type and CD4+CD45RO+cells act as TH2 type in an antigen-independent manner. We also observed strong T-bet activation in CD8+ T cells in both subsets. Signal transmission and activation through TNFR family members, such as CD27-mediated co-receptor signaling may increase Th1 effector functions and be possibly beneficial in immunity against tumors and viral infections.