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The objective of the present paper was to link reach-scale streambed reactive uptake of dissolved organic carbon (DOC) and dissolved oxygen (DO) to subsurface flow paths in an alpine stream (Oberer Seebach (OSB)). The topography adjacent to the stream channel largely determined flow paths, with shallow hillslope groundwater flowing beneath the stream and entering the alluvial groundwater at the opposite bank. As computed from hydrometric data, OSB consistently lost stream water to groundwater with fluxes out of the stream averaging 943 ± 47 and 664 ± 45 L m−2 h−1 at low (Q < 600 L s−1) and high (Q > 600 L s−1) flow, respectively. Hydrometric segregation of streambed fluxes and physicochemical mixing analysis indicated that stream water was the major input component to the streambed with average contributions of 70–80% to the hyporheic zone (i.e., the subsurface zone where shallow groundwater and stream water mix). Surface water was also the major source of DOC with 0.512 ± 0.043 mg C m−2 h−1 to the streambed. The DOC flux from shallow riparian groundwater was lower (0.309 ± 0.071 mg C m−2 h−1) and peaked in autumn with 1.011 mg C m−2 h−1. I computed the relative proportion of downstream discharge through the streambed as the ratio of the downstream length (Ssw) a stream water parcel travels before entering the streambed to the downstream length (Shyp) a streambed water parcel travels before returning to the stream water. The relative streambed DOC retention efficiency, calculated as (input-output)/input of interstitial DOC, correlated with the proportion (Ssw/Shyp) of downstream discharge (r2 = 0.76, p = 0.006). Also, did the streambed metabolism (calculated as DO uptake from mass balance) decrease with low subsurface downstream routing, whereas elevated downstream discharge through the streambed stimulated DO uptake (r2 = 0.69, p = 0.019)? Despite the very short DOC turnover times (∼0.05 days, calculated as mean standing stock/annual input) within the streambed, the latter constitutes a net sink of DOC (∼14 mg C m−2 h−1). Along with high standing stocks of sediment associated particulate organic carbon, these results suggest microbial biofilms as the major retention and storage site of DOC in an alpine stream where large hydrologic exchange controls DOC fluxes.