13C-NMR spectroscopy was used to determine the level of cytoplasmic malate in maize root tips that exhibited different rates of malate synthesis. Intracellular malate was 13C-labeled at carbons 1 and 4 by perfusing root tips with 5 nM H13CO3-. This labeling reflects the activities of phosphoenolpyruvate carboxylase and malate dehydrogenase (production of [4-13C]malate), and fumarase (scrambling of 13C-label between C1 and C4 of malate). In vivo 13C-NMR spectra contained a clearly resolved resonance from cytoplasmic [4-13C]malate, while the resonance from cytoplasmic [1-13C]malate overlapped with others. After 90 min of H13CO3- treatment, 13C-labeling of organic acid pools had reached steady-state. Thereafter, the ratios [13C]malate/[12C + 13C]malate and [1-13C]malate/[4-13C]malate in tissue extracts remained constant; evidence is presented that these ratios were the same for both cytoplasmic and total cellular malate. Hence, the intensity of the cytoplasmic [4-13C]malate signal was proportional to the amount of cytoplasmic malate in root tips. Potassium sulfate stimulate malate synthesis in maize root tips, relative to root tips perfused with HCO3- alone; total cellular malate doubled after approx. 1 h of 5 mM K2SO4-treatment. Cytoplasmic malate increased from approx. 3.5 mM to approx. 7.5 mM within 45 min of the onset of K2SO4-treatment, declining slightly thereafter. The possible effects of these changing cytoplasmic malate concentration on the enzymes involved in malate metabolism are discussed.