Vacuoles were isolated from leaves of two lettuce (Lactuca sativa L.) genotypes that differed significantly in their nitrate accumulation. The rate of nitrate uptake into the vacuoles did not differ between the genotypes, but the malate-uptake rate did. Fitting a Michaelis-Menten equation, with or without the addition of a linear term, showed that the rates of malate and nitrate uptake can be best described by saturation kinetics for both genotypes. Malate transport across the tonoplast showed a K m value of approx. 40 mM, while the K m value for nitrate uptake was approx. 5 mM. Both malate and nitrate uptake were greatly stimulated by ATP, but not by pyrophosphate. Valinomycin considerably blocked both malate and nitrate uptake while nigericin only slightly affected the rate of malate uptake and had no effect on the nitrate-uptake rate. This indicates that both nitrate and malate transport are driven by the membrane potential, while the pH gradient may play a minor role in malate transport only. The presence of nitrate in the incubation medium inhibited malate uptake (2 mM nitrate caused an inhibition of approx. 50%). In contrast to this, the presence of malate in the incubation medium did not inhibit nitrate uptake. Endogenous nitrate did not affect malate uptake. Thus, we did not find genotypic differences in the uptake pattern which could explain the variation in nitrate accumulation. The possible reason for differences in nitrate accumulation in vivo is discussed.