Using the Fura-2 method we investigated a possible direct action of testosterone on cytosolic free calcium of splenic T cells isolated from female C57BL/10 mice. Testosterone at physiological concentrations of 1-10 nM induces an increase in [Ca2+]i within seconds, which is due to Ca2+ influx and not to Ca2+ release from intracellular stores. In contrast, estradiol induces both Ca2+ influx and Ca2+ release. The testosterone-induced Ca2+ influx is mediated by Ni2+-blockable channels and is not inhibited by cyproterone, a blocker of the classical androgen receptor. Ca2+ influx can also be induced by testosterone conjugated to BSA which is impermeable to the plasma membrane. These data indicate a novel mode of direct action of testosterone on T cells which is not mediated through the classical androgen receptor response, but through unconventional plasma membrane receptors.