To quantify the contribution of hyporheic community respiration to whole running-water ecosystem respiration in a cultural landscape setting, we studied the vertical hydraulic exchange in riffle–pool sequences of the River Lahn (Germany). We used flow through curves from four tracer experiments to estimate flow velocities in the surface and subsurface water. Generally, vertical exchange velocities were higher in riffle sections and a high temporal variability was observed (range of values 0.11–1.08 m day⁻¹). We then used (1) the exchange velocities and (2) time series of dissolved oxygen concentration in surface and subsurface water to calculate hyporheic respiration. Hyporheic respiration was estimated in a range of 10–50 mg O₂ m⁻³ day⁻¹ for the upper sediment layer (first 20 cm). It was much lower in the deeper sediment layer (20–40 cm), ranging from 0 to 10 mg O₂ m⁻³ day⁻¹ (volumes are volumes of interstitial water; the average porosity was 20%). We determined primary production and respiration of the biofilm growing on the sediment by modelling dissolved oxygen concentration time series for a 2,450 m long stream reach (dissolved oxygen concentrations with diurnal variations from 8 to 16 mg L⁻¹). Modelled respiration rates ranged from 2 to 21 g O₂ m² day⁻¹. All information was integrated in a system analysis with numerical simulations of respiration with and without sediments. Results indicated that hyporheic respiration accounted for 6 to 14% of whole ecosystem respiration. These values are much lower than in other whole system respiration studies on more oligotrophic river systems.