We monitored water nutrient concentrations, periphyton biomass, and periphyton cellular nutrient concentrations in run and riffle habitats at sites representative of headwater, mid-catchment and lowland valley segments of a grassland river for two years, and community composition on four occasions during this period, to determine how these communities varied spatially and temporally as a function of downstream changes in hydraulic conditions, flood disturbance regimes and enrichment. Predictions of community biomass and structure were tested under a proposed habitat matrix conceptual model for periphyton. Discharge increased and valley segment slope decreased in a downstream direction as expected from geomophic models of catchment processes. However, site-specific depths and velocites did not change systematically down the river. Water nutrient concentrations suggested a change from nitrogen limitation of primary production at the headwater site to phosphorus limitation at the lowland site. This was associated with a downstream increase in stream nitrate concentrations, but not phosphorus. However, cellular nutrient concentrations and instream nutrient bioassays suggested either N limitation, or weak N and P limitation, at all sites. In runs, mean monthly chlorophyll-a did not vary significantly (P>0.05) among the three sites, but it did in riffles. The differences in community biomass between run and riffle habitats were more significant than differences among sites. Periphyton community structure in the runs was similar among valley segments during a period of frequent flood disturbances, but during a period of infrequent disturbances communities in the headwater sites were dominated by filamentous cyanobacteria whereas in the mid-catchment and lowland sites they were dominated by nitrogen-fixing cyanobacteria and diatoms. Major differences in community structure occurred in riffles among the sites. Riffle communities at the headwater site were dominated by filamentous cyanobacteria and diatoms, whereas at the mid-catchment and lowland sites filamentous green algae and diatoms were dominant, possible a result of increased nitrate concentrations progressing downstream. Overall, a downstream gradient in nitrate enrichment appeared to control biomass and community composition of the riffle communities, but not the run communities. Strong grazer activity in runs during more hydrologically stable periods at the lowland site appeared to override any response to nutrients and maintained biomass at low levels. Periphyton biomass and community composition was predicted more accurately from the habitat matrix conceptual model based on local habitat factors than by expected downstream gradients in hydraulic conditions and enrichment.