While numerous studies have examined N dynamics along a river continuum, few have specifically examined the role of biofilms. Nitrogen dynamics and microbial community structure were determined on biofilms at six sites along a 120km stretch of the lowland Ovens River, South Eastern Australia using artificial Substrates. Terminal restriction fragment length polymorphism (T-RFLP), chlorophyll a and protein analyses were used to assess biofilm microbial community composition. N dynamics was determined on the biofilms using the acetylene (C2H2) block technique and assessing changes in NH4+, NOx, and N2O. Unlike microbial community structure, N dynamics were spatially heterogeneous. Nitrification, determined from the difference in accumulation of NH4+ before and after addition of C2H2, occurred mostly in the upper sites with rates up to 1.4 x 10(-5) mol m(-2) h(-1). The highest rates of denitrification occurred in the mid-reaches of the river (with rates up to 1 X 10(-5) mol m(-2) h(-1)) but denitrification was not detected in the lower reaches. At the very most, only 50% of the observed uptake of NOx by the biofilms following addition of C2H2 could be accounted for by denitrification.