Nitrate loads from the upper Mississippi River basin contribute substantially to the Gulf Hypoxia problem, and nitrates in the St. Croix River, an upper Mississippi River tributary, have increased steadily since the 1970s.  We used stable isotopes to help identify sources of nitrogen in the lower St. Croix River.  Water samples were collected for analysis of nitrate concentration and nitrate ¹⁵N from seven mainstem sites, five tributaries, and two wastewater treatment plants from May through October, 2008, capturing a range of hydrologic conditions.  Additionally, seston and zebra mussels were collected (July-October and May-October, respectively) from mainstem sites for analysis of ¹⁵N.  Mean mainstem nitrate concentrations increased from upstream to downstream sites (0.15-0.39 mg N/l), with higher concentrations found in tributaries (0.79-5.65 mg N/l) and treated wastewater (5.61-14.58 mg N/l).  d¹⁵N values in seston varied from approximately 3 to 12; preliminary d¹⁵N data for zebra mussel tissue were less variable, ranging from 7 to 11.  Among mainstem sites, d¹⁵N signatures in seston and zebra mussels were strongly correlated with nitrate concentrations (r²=0.84 and r²=0.78, respectively), suggesting that animal-based nitrogen sources (e.g., wastewater inputs, tributary dairy operations) contribute to the pattern of increasing nitrate concentrations in the lower St. Croix River.