We analyzed ecoenzyme activities related to organic matter processing in 1879 streams and rivers across the continental U.S. as part of the USEPA's National Rivers and Streams Assessment. Ecoenzymatic stoichiometry was used to construct models for carbon use efficiency (CUE) and decomposition (−k). Microbial respiration (Rm) was estimated from sediment organic carbon stocks, CUE, and −k. The streams and rivers were classified by size (headwaters: 1st-order; streams: 2nd–3rd order; small rivers: 4th–5th order; big rivers 6th–7th order; and great rivers ≥ 8th order) and condition class (least, intermediate, and most disturbed), and grouped into nine ecoregions. There were ecoregion, stream size, and condition class effects for CUE, −k, and Rm, with Rm increasing from eastern ecoregions through the plains to the western ecoregions. CUE, −k, and Rm decreased with increasing streams size and increased with increasing disturbance. Rm, CUE, and −k were correlated with water and sediment chemistry; CUE and −k were also correlated with stream bed fine sediments; and CUE was further correlated with catchment land cover. Rm was extrapolated to ecoregional and national scales, and the results suggest that microbial assemblages account for 12% of the total CO2 outgassing, and nearly 50% of the aquatic metabolism C losses, from U.S. streams and rivers. Cumulative respiratory C losses increased from headwaters to small streams, then decreased with increasing stream size. This U-shaped respiration curve was not evident when streams were viewed by disturbance classes, suggesting that anthropogenic disturbances mask the expected organic matter processing signature of the river continuum.