The stimulation of different cell types with growth factors is often accompanied by a rapid intracellular alkalinization. By using mitogenic lectins, cluster of differentiation (CD)2 and CD3 mAb, as stimuli, we studied early changes of the intracellular pH in the activation process of resting human PBL. We found increases in free cytoplasmic Ca2+ levels and DNA synthesis but no intracellular alkalinization in the early activation phase upon stimulation with the mitogenic lectins, Con A, and PHA. Similarly stimulation with CD3 mAb led in most instances to no detectable pH shifts. Only in 7 out of 30 experiments was CD3 mAb-induced alkalinization observed. In contrast, stimulation with mitogenic combinations of anti-CD2 mAb led in all instances to rapid and clear-cut intracellular pH shifts very similar to those observed upon stimulation with PMA. In medium lacking sodium bicarbonate the intracellular alkalinization via the CD2 structure could be blocked by the amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA), which indicates that this increase in pH is mediated by the amiloride-sensitive Na+/H+ antiporter. Blockade of this antiporter had no negative effect, however, on T cell proliferation as measured by thymidine incorporation. In contrast, significantly enhanced proliferation rates were observed after stimulation with mitogenic combinations of anti-CD2 antibodies in the presence of MIA. No such effect of MIA could be observed in lectin induced T cell stimulation. These findings indicate that stimulation of the Na+/H+ antiporter via the CD2 structure is neither a prerequisite for T cell proliferation nor does it promote T cell growth. It rather seems to function in a regulatory role. In its absence, superinduction of proliferation can be achieved.