Nitrogen deficiency tolerance is necessary to breed for wheat varieties adapted to low input management systems. The aim of this study was to identify root proteins differentially expressed in response to a nitrogen stress. Two wheat varieties were experimented at low (0.5mM nitrate) and at high (3.0mM nitrate) nitrogen levels. After 8 weeks, root samples were taken to assess enzyme activities and to perform two dimensional gel electrophoresis. A total of 860 root protein spots were qualitatively or quantitatively analysed. A significant variety effect existed for 74 spots, while a significant nitrate supply-dependent variation was observed for 56 spots. Moreover 20 spots showed a significant variety × N-supply effect. Correlations were made between physiological parameters (nitrogen content and enzyme activities) and the intensity of variable spots. Positive correlations were observed for nitrate reductase and glutamate dehydrogenase activities and the intensity of a number of root proteins. This approach may be a way to identify new structural or regulatory proteins involved in the response to the level of nitrogen fertilisation in different wheat varieties.