The intracellular pH of isolated, photosynthetically active mesophyll cells of Asparagus sprengeri Regel has been determined, in the light and dark, by the distribution of the weak acid 5,5-dimethyl-[2-(14)C]oxazolidine-2,4-dione ([(14)C]DMO) between the cells and the liquid medium. [(14)C]DMO was taken up rapidly, reaching equilibrium in 7-10 min of incubation, but was not metabolized by the cells, and intracellular binding of the compound was minimal. The intracellular pH, measured at saturating light fluence and 1.5 mM sodium bicarbonate, was found to remain relatively constant at 6.95-7.21 over the external pH range of 5.5-7.2. Illumination of the cells increased the intracellular pH compared to dark controls. The pH of the cytoplasm, excluding and including the chloroplasts ("cytoplasmic" and "bulk cytoplasmic", respectively) was calculated from the experimentally derived intracellular [(14)C]DMO concentration and estimates of the vacuolar, chloroplastic and cytoplasmic volumes. The calculated cytoplasmic pH was similar in the light and dark, being 7.75 and 7.74, respectively, while the calculated pH of bulk cytoplasm was 7.85 in the light and 7.49 in the dark. Theoretical analysis indicated that intracellular pH is a good indicator of changes in the bulk cytoplasmic pH but insensitive to changes in vacuolar pH. The external pH optimum for photosynthesis (O2 evolution) of isolated Asparagus cells was pH 7.2. At pH 8.0 photosynthesis was inhibited by 30% and at pH 5.25 by 45%. Inhibition at alkaline pH may be the result of a decrease in the pH gradient between the cells and the medium, causing CO2 limitation in the cell. At acid pH, decrease in internal pH caused by substantial accumulation of inorganic carbon may account for the loss in photosynthetic activity.