Abstract In general, respiration (RS) is highly correlated with nitrogen concentration (N) in plant organs, including roots, which exhibit a positive N–RS relationship. Less is known, however, about the relationship between N and RS in roots of different branch orders within an individual tree along a vertical soil profile; this is especially true in trees with contrasting life strategies, such as pioneer Scots pine (Pinus sylvestris L.) vs mid-successional sessile oak (Quercus petraea Liebl.). In the present research, the impact of root branch order, as represented by those with absorptive vs transporting ability, and soil genetic horizon on root N, RS and the N–RS relationship was examined. Mean RS and total N concentration differed significantly among root branch orders and was significantly higher in absorptive roots than in transporting roots. The soil genetic horizon differentially affected root RS in Scots pine vs sessile oak. The genetic horizon mostly affected RS in absorptive roots of Scots pine and transporting roots in sessile oak. Root N was the highest in absorptive roots and most affected by soil genetic horizon in both tree species. Root N was not correlated with soil N, although N levels were higher in roots growing in fertile soil genetic horizons. Overall, RS in different root branch orders was positively correlated with N in both species. The N–RS relationship in roots, pooled by soil genetic horizon, was significant in both species, but was only significant in sessile oak when roots were pooled by root branch order. In both tree species, a significant interaction was found between the soil genetic horizon and root branch order with root function; however, species-specific responses were found. Both root N, which was unaffected by soil N, and the positive N–RS relationship consistently observed in different genetic horizons suggest that root function prevails over environmental factors, such as soil genetic horizon.