Creightons Creek, a rural stream in southeastern Australia, has been severely degraded by large-scale sedimentation of sand in the streambed. Our paper highlights the use of whole-stream metabolism measurements, fluorescein diacetate hydrolysis studies, and in situ enzyme mapping to examine the effect of sediment instability on benthic and hyporheic metabolism across seasonal and flow variations in Creightons Creek. Median gross primary production (GPP) and ecosystem respiration (ER₂₄) rates, determined over a 20-mo period with the whole-stream single-station diurnal O₂ change method, ranged between 0 and 0.5 g O₂ m⁻² d⁻¹ and 0.6 and 3.7 g O₂ m⁻² d⁻¹, respectively. These values gave photosynthesis/respiration (P/R) ratios between 0.00 and 0.41, indicative of a heterotrophic system. Creightons Creek was expected to support higher GPP because it has an open canopy, clear water, warm temperatures, and sufficient nutrients. However, this once-clay-bottomed stream now has an abrasive benthic layer of sand that moves continuously, even at low flows, and this layer has inhibited the growth of benthic primary producers (0–2 mg/m² of chlorophyll a). During high flows, when scour and abrasion from the bed movement were at a maximum, the highest relative enzyme activity was located in the hyporheic zone at a depth of 7 to 12 cm, where the sediment was more stable. During low flows, the highest enzyme activity occurred in the upper 4 cm of the sediment profile. Our study shows that the instability of the sediment bed is a major factor determining the rates and locations of metabolism.