Purified pertussis toxin (PPT) is a potent mitogen for human T lymphocytes and is shown to cause rapid calcium mobilization in resting T cells, a T-cell line and CD3- lymphocytes with natural killer (NK) activity. In resting T cells the PPT activation is associated with cytoplasmic alkalinization. A similar rise in intracellular free calcium ([Ca2+]i) and cytoplasmic alkalinization is observed with activation through the antigen receptor complex. The effect of PPT is unlikely to be mediated through this pathway, however, as it can mobilize calcium in lymphocytes that do not express the CD3-Ti complex. In contrast to several other cell types, re-incubation of resting human T cells with PPT, up to a dose of 100 ng/ml for 2 hr does not block subsequent agonist-induced calcium mobilization dependent on G protein-mediated phospholipase C activation. Mitogenic doses of PPT cause a modest reduction in subsequent agonist responses, but this is likely to be due to a post-activation refractory state rather than G protein inactivation.