The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.