Monoclonal antibodies recognizing CD27, a member of the nerve growth factor receptor family, have been observed to enhance or inhibit PHA- or CD3 mAb-stimulated T cell proliferation. In this study, we investigate the effects of CD27 stimulation on the proliferation and cytokine production of T cells stimulated simultaneously through CD3, CD2, or CD28. Here we report that simultaneous crosslinking of CD27 plus CD28 results in T cell proliferation and in the production of IL-2, IL-4, IL-10, and IFN-gamma. CD27 plus CD28 stimulation thus introduces a novel Ag-independent pathway of T cell activation. We further show that all major T cell subsets (CD4+, CD8+, CD4+CD45RA+, or CD4+CD45RO+ T cells) respond to CD27 plus CD28-mediated costimulation. Synergistic effects on T cell stimulation are also found when CD27 is crosslinked on cells stimulated simultaneously through CD3 or CD2. In further experiments we show that crosslinking of CD27 elevates cytoplasmic free Ca2+ levels. Finally, both the CD3 plus CD27 and the CD28 plus CD27-stimulated T cell proliferation is sensitive to inhibition by CsA. Taken together, these data emphasize the importance of CD27 stimulation in the context of simultaneously received signals through CD3, CD2 or, most importantly, through CD28. Furthermore, signaling through CD27 appears to involve Ca(2+)-dependent mechanisms sensitive to CsA.