- The effects of substratum geometry and overlying velocity on nitrate use by periphyton were assessed. Periphyton was cultivated at an average current velocity of 0.5 cm s(-1) in laboratory mesocosms (120 cm long, 60 cm wide) on polyethylene nets of three different geometries, "1-lay er", "3-layer", and "bedform" structures, overlaying a thin bed of sand. Bulk nitrate use was then measured as the reduction of nitrate concentration in the overlying water under average velocities of 0.005, 0.05, and 0.5 cm s(-1). Periphyton structural characteristics were quantified as algal/bacterial biomass, algal species composition, and bacterial densities. Accrual of microbial biomass increased monotonically with increasing benthic net surface area, with upper sections of structures supporting the highest biomass. Maximum rates of nitrate removal were measured in the bedform geometry at intermediate velocity (173 mg NO3-N m(-2) d(-1)), and the lowest was measured with 1-layer geometry at the fastest velocity (11 mg NO3-N m(-2) d(-1)). Oxygen microprofiles within biofilms demonstrated that hydrodynamic conditions and benthic structure both play a key role in the regulation of microbial processing of nitrate delivered from the water column by promotion of denitrification in downstream sections of bedform substrata. Interactions between hydrodynamic conditions and substratum geometry are expected to regulate microbial activity in all surficial natural and engineered environments and must be parameterized to forecast long-term average biochemical transformation rates in rivers and other dynamic aquatic systems.