Stress-activated protein kinases (SAPK) are stimulated by a variety of agents and conditions that also activate the Na+/H+ exchanger (NHE). Activation of the exchanger results in a rapid increase in intracellular pH (pHi), raising the possibility that cytosolic alkalinization may contribute to SAPK activation. This hypothesis was tested by manipulating the pHi of U937 cells using permeant weak bases. Three different bases increased pHi and caused a 4-12-fold increase in SAPK activity with a time course that paralleled intracellular alkalinization. p38, a related stress kinase, was also stimulated by the weak bases. Stimulation of the stress kinases was not accompanied by changes in cytosolic free calcium nor was the activation of SAPK achieved when calcium was elevated by thapsigargin or calcium ionophores. Weak bases not only alter the pH of the cytosol but also alkalinize endomembrane compartments such as endosomes and lysosomes. However, the latter do not appear to mediate the stimulation of SAPK, since neither bafilomycin A1 nor desipramine, agents that neutralize acidic endomembrane compartments, activated the kinase. Because hyperosmolarity acutely activates the NHE, we considered whether the resulting cytosolic alkalinization mediates the activation of SAPK upon cell shrinkage. The addition of amiloride or the omission of Na+, which were verified to inhibit NHE, did not prevent the osmotically induced activation of SAPK. We conclude that cytosolic alkalinization increases the activity of SAPK and p38 by a calcium-independent mechanism that does not involve acidic intracellular organelles. In addition, even though cell shrinkage is accompanied by alkalinization due to the activation of NHE, the increased pHi is not the main cause of the observed stimulation of SAPK upon hyperosmotic challenge.