The use of smart tracers to study hydrologic systems is becoming more widespread. Smart tracers are compounds that irreversibly react in the presence of a process or condition under investigation. Resazurin (Raz) is a smart tracer that undergoes an irreversible reduction to resorufin (Rru) in the presence of cellular metabolic activity. We quantified the relationship between the transformation of Raz and aerobic bacterial respiration in pure culture experiments using two obligate aerobes and two facultative anaerobes, and in colonized surface and shallow (<10 cm) hyporheic sediments using reach-scale experiments. We found that the transformation of Raz to Rru was nearly perfectly (minr2 = 0.986), positively correlated with aerobic microbial respiration in all experiments. These results suggest that Raz can be used as a surrogate to measure respiration in situ and in vivoat different spatial scales, thus providing an alternative to investigate mechanistic controls of solute transport and stream metabolism on nutrient processing. Lastly, a comparison of respiration and mass-transfer rates in streams suggests that field-scale respiration is controlled by the slower of respiration and mass transfer, highlighting the need to understand both biogeochemistry and physics in stream ecosystems.