High concentrations of phenolics have been shown to play a role in plant resistance to pathogens. One way to obtain increased phenolic concentrations in plant tissues is to limit mineral nitrogen (N) availability; however, over long periods, this treatment will have a negative effect on plant growth. The aim of our study was to determine the effect of repeated short-term N limitations on plant growth and phenolic metabolism in leaves. Tomato plants (Solanum lycopersicum, cv. Pixie) were subjected to two successive 10-day N-limitation periods (0.15 mM NO3-, 0.01 mM NH4+), followed by periods of full nutrient supply (15 mM NO3-, 1.2 mM NH4+). Additionally, other plants were subjected to either of these two limitation periods, and a set of control plants was given a full nutrient supply during the entire period. The phenolic metabolism was monitored by measuring the leaf concentrations of chlorogenic acid, three flavonol glycosides (quercetin and kaempferol derivatives) and two major anthocyanins, together with the expression of eight structural genes and three transcription factors of the phenylpropanoid pathway. The relative growth rate of the plants decreased during the N-limitation periods but was restored as soon as N was resupplied. Each N-limitation period resulted in an up-regulation of the phenolic biosynthetic pathway, as demonstrated by an increase in the leaf phenolic concentration and an up-regulation of the related genes. The genes in the phenolic pathway were down-regulated immediately when N was resupplied; however, the leaf concentrations of several phenolics, particularly flavonol glycosides, were maintained at significantly higher levels than in the control plants for up to 17 days after the end of the first limitation. The amplitude of the increase in leaf phenolic concentration did not depend on the number of N-limitation periods to which the plant was subjected, which indicates that the plants did not acclimate to nitrogen limitation. Successive N-limitation periods resulted in additive increases in flavonol glycoside concentrations. (C) 2012 Elsevier Ltd. All rights reserved.