PGE₂-mediated suppression of T cell proliferation during sepsis could result from altered Ca²⁺ signaling. The present study evaluated the effects of PGE₂ on Ca²⁺ release from intracellular stores and its influx through the plasma membrane in splenic T cells from Sprague-Dawley rats. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) responses in individual T cells were assessed using the Ca²⁺ imaging technique, and the release of Ca²⁺ from intracellular stores and Ca²⁺ influx were spectrofluorometrically quantified in T cell suspensions. Under unstimulated conditions, nearly 85% of T cells exhibited [Ca²⁺]_i 50 nM. After stimulation with concanavalin A (Con A), an increase in [Ca²⁺]_i was recorded in ~60% of the cells. The pretreatment of T cells with PGE₂ had no apparent effect on [Ca²⁺]_i in resting cells; it significantly suppressed the Con A-induced increase in [Ca²⁺]_i in all of the Con A-responsive cells. Ca²⁺ release from the intracellular stores contributed to the early spike in [Ca²⁺]_i, and the late phase of elevation in [Ca²⁺]_i was dependent on Ca²⁺ influx through the plasma membrane. Our data suggest that PGE₂ causes an overall suppression of the Con A-induced [Ca²⁺]_i elevation in T cells via inhibiting both Ca²⁺ influx and its release from the intracellular stores.