Nitrogen distribution within the Upper Mississippi River (UMR) is both spatially and temporally heterogeneous.  Past work indicated sites highly connected to the river’s main channel had higher dissolved inorganic nitrogen (DIN), while less connected sites had lower DIN and low nitrogen to phosphorus (N:P) ratios.  We conducted biweekly acetylene reduction assays at 6 sites, from May to September, to determine the temporal and spatial extent of N-fixation.  In late July, the least connected site had the highest N-fixation rates (216 μM C₂H₄ · m^-2 · h^-1), while low rates (20 μM C₂H₄ · m^-2 · h^-1) were observed in the main channel.  The onset of fixation was marked by declining N:P ratios prior to the chlorophyll a maxima.  No threshold N:P ratio consistently predicted the onset of fixation.  N-fixation is thought to be a summer phenomenon when DIN and N:P ratios are low favoring the cyanobacteria blooms.  Our research supported this contention, but also indicated that N-fixation had already occurred and was declining by late May and June in the two least connected sites.  Ironically, this reach of the UMR transports ~80,000 terragrams of nitrogen per year and can still be nitrogen limited at certain times and places for some autotrophic taxa.