The effect of elevating cytoplasmic Ca" ([Ca2+ ] i) on the intracellular pH (pH i) ofthymic lymphocytes was investigated . In Nay'-containing media, treatment of the cells with ionomycin, a divalent cation ionophore, induced a moderate cytoplasmic alkalinization . In the presence of amiloride or in Na'free media, an acidification was observed . This acidification is at least partly due to H' (equivalent) uptake in response to membrane hyperpolarization since : (a) it was enhanced by pretreatment with conductive protonophores, (b) it could be mimicked by valinomycin, and (c) it was decreased by depolarization with K' or gramicidin . In addition, activation of metabolic H+ production also contributes to the acidification . The alkalinization is due to Na'/H' exchange inasmuch as it is Na' dependent, amiloride sensitive, and accompanied by H+ efflux and net Na' gain . A shift in the pHi dependence underlies the activation of the antiport . The effect of [Ca"] ; on Na'/H' exchange was not associated with redistribution of protein kinase C and was also observed in cells previously depleted of this enzyme . Treatment with ionomycin induced significant cell shrinking . Prevention of shrinking largely eliminated the activation of the antiport . Moreover, a comparable shrinking produced by hypertonic media also activated the antiport . It is concluded that stimulation of Na'/H' exchange by elevation of [Ca"] ; is due, at least in part, to cell shrinking and does not require stimulation of protein kinase C .