Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco₂ (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH_i). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO₂-HCO₃, propionic acid-propionate, or NH₃-NH₄⁺. In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH_i (pH_i)  0.38; exponential time constant ()  120 s]. In the presence of 5% CO₂, 2T caused a biphasic pH_i response: a rapid increase (pH_i  0.10,   15 s) followed by a slower pH_i increase. Identical rapid pH_i increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH_i decrease (pH_i  0.21,   10 s) in the presence of NH₃ (20 mM). Thus osmotic cell shrinkage caused rapid pH_i changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO₂ or propionic acid) vs. a weak base buffer (contraction acidosis with NH₃). Graded pH_i were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant pH_i. The rapid pH_i responses to 2T were insensitive to inhibitors of membrane H⁺ transport (ethylisopropylamiloride and bafilomycin A₁). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).