Sequential stimulation and washout procedures were employed to examine the kinetics and reversibility of pharmacologically manipulated second messenger signals mediating phenotypic changes and proliferative activation of resting human T lymphocytes. Phorbol dibutyrate (PDBu) was used to stimulate protein kinase C (Ca2+/phospholipid-dependent enzyme) while ionomycin was used to manipulate intracellular Ca2+ levels. Stimulation by PDBu alone induced phosphorylation of several endogenous substrates and altered expression of phenotypic markers, downregulating expression of CD4 and CD3 while increasing expression of CD2 and the interleukin 2 (IL-2) receptor. Stimulation with ionomycin alone caused an increase in intracellular Ca2+ levels but did not induce proliferation or cause major changes in the expression of phenotypic markers (CD2, CD3, CD4, CD8, IL-2, and transferrin receptors). Analysis of endogenous PDBu stimulated phosphosubstrates indicated that some substrates (pp92, pp82, pp55) underwent dephosphorylation, returning to base-line levels following PDBu removal while others (pp61, pp65) showed only partial dephosphorylation, while one (pp28) remained phosphorylated. Washing ionomycin-stimulated cells resulted in an approximately 75% reduction of intracellular Ca2+. Ionomycin exposure did not alter the affinity (KD = 22.3 +/- 7.4 nM) or number of receptors (53,497 +/- 8,291 receptors/cell) for [3H]PDBu. These data suggest that signals induced by PDBu or ionomycin are reversible following removal of the stimulating agents with respect to proliferative activation of T lymphocytes. Furthermore, a transcriptional mechanism regulating the production of IL-2 mRNA requires simultaneous activation of protein kinase C and elevation of intracellular Ca2+.