AbstractMost measurements of respiration rates in large tropical rivers do not account for the influence of river flow conditions on microbial activity. We developed a ship‐board spinning incubation system for measuring O2 drawdown under different rotation velocities and deployed the system along the lower Amazon River during four hydrologic periods. Average respiration rates in incubation chambers rotated at 0.22 m s−1 and 0.66 m s−1 were 1.4 and 2.4 times higher than stationary chambers, respectively. On average, depth‐integrated respiration rates in chambers spun at 0.22 m s−1 and 0.66 m s−1 accounted for 64% ± 22% and 104% ± 36% of CO2 outgassing rates, respectively, in mainstem sites. Continuous measurements of in situ pCO2 were also made along with cross‐channel profiles of river velocity. A positive correlation between river velocity and pCO2 was observed along the lower river (r2 = 0.67–0.96) and throughout a tidal cycle.