We examined total nitrogen to total phosphorus (TN:TP) ratios and limitation of periphyton biomass in four mid-sized (5^th to 7^th order) rivers in southeast Idaho.  TN:TP showed similar seasonal patterns, with higher ratios occurring from November through February, when biological activity is lowest.  Generally, within rivers, TN:TP increased downstream during periods of low biological activity, suggesting disproportional increases of TN relative to TP.  Reaches varied in proximity to dams, and for most sites and dates, TN:TP below dams was elevated relative to sites above dams.  Most of this increase was due to storage of P in reservoirs, leading to reduced TP concentrations downstream.  Nutrient diffusing substrate bioassays were conducted at 11 sites (2 to 5 sampling events) from August 2005 to October 2006 within these rivers.  Overall, results from bioassays indicate that N most often limited periphyton biomass, although frequently biomass was not nutrient limited.  When N limitation was observed, TN:TP ranged from 0.66 to 75.71.  In 10 of 17 events lower TN:TP ratios (<20) and TP concentrations (<0.15 mg/L) coincided with bioassay responses.  These responses always indicated N limitation, but co-limitation by P also occurred. This work suggests that TN:TP is not a consistent predictor of periphyton nutrient limitation.