Aims Intercropping legumes and non-legumes may affect the root growth of both components in the mixture, and the non-legume is known to be strongly favored by increasing nitrogen (N) supply. The knowledge of how root systems affect the growth of the individual species is useful for understanding the interactions in intercrops as well as for planning cover cropping strategies. The aim of this work was (i) to determine if different levels of N in the topsoil influence root depth (RD) and intensity of barley and vetch as sole crops or as an intercropped mixture and (ii) to test if the choice of a mixture or the N availability in the topsoil will influence the N uptake by deep roots.Methods In this study, we combined rhizotron studies with root extraction and species identification by microscopy with studies of growth, N uptake and 15N uptake from deeper soil layers, for studying the root interactions of root growth and N foraging for barley (Hordeum vulgare L.) and vetch (Vicia sativa L.), frequently grown in mixtures as cover crops. N was added at 0 (N0), 50 (N1) and 150 (N2) kg N ha−1. The roots discrimination relying on the anatomical and morphological differences observed between dicots and monocots proved to be a reliable method providing valuable data for the analysis.Important Findings The intercrop and the barley attained slightly higher root intensity (RI) and RD than the vetch, with values around 150 crosses m−1 and 1.4 m, respectively, compared to 50 crosses m−1 and 0.9 m for the vetch. At deep soil layers, intercropping showed slightly larger RI values compared to the sole-cropped barley. The barley and the intercropping had larger root length density (RLD) values (200–600 m m−3) than the vetch (25–130) at 0.8–1.2 m depth. The topsoil N supply did not show a clear effect on the RI, RD or RLD; however, increasing topsoil N favored the proliferation of vetch roots in the intercropping at deep soil layers, with the barley:vetch root ratio ranging from 25 at N0 to 5 at N2. The N uptake of the barley was enhanced in the intercropping at the expense of the vetch (from ~100mg plant−1 to 200). The intercropped barley roots took up more labeled nitrogen (0.6mg 15N plant−1) than the sole-cropped barley roots (0.3mg 15N plant−1) from deep layers.