The effects of arachidonic acid (AA) and the lectin mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA), on [Ca2+], and pHi in rat thymocytes have been studied by using the intracellular fluorescent probes, Fura-2 and BCECF. It was revealed that exogenous AA (3 μM), in addition to the well-known changes in basal [Ca2+]i and pHi, also caused a complete blockade of [Ca2+], and pH, signals induced by Con A(10μg/ml) and PHA (10 μg/ml). In contrast, exposure of thymocytes to mitogens did not prevent the AA-induced increase in [Ca2+]i, and decrease in pHi, In experiments with sodium propionate, the similarity between AA action and EIPA (ethylisopropylamiloride), an inhibitor of Na+/H+ exchangers, was revealed. It is proposed that the inhibitory effect of AA on mitogen-induced lymphocyte proliferation is due primarily to the blockade of transmembrane [Ca2+]i, and pHi signals, associated with a sustained cytosolic acidification.