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The concentration of dissolved organic nitrogen (DON) is increasing in many northern hemisphere lakes, yet its use by phytoplankton and fate in the environment seldom have been quantified. We conducted 1 week, in situ, microcosm incubations across 25 lakes in northeastern North America to understand how DON, dissolved inorganic nitrogen (DIN), and dissolved inorganic phosphorus (P) affected phytoplankton biomass. In addition, we tested whether lakes were limited by single macronutrients (N or P) or colimited by both. Phytoplankton biomass in 80% of lakes responded similarly to DON and DIN additions. Of the lakes where N form produced differential responses, the majority of phytoplankton communities exhibited greater biomass accumulation with DON than DIN. Colimitation was the most common type of nutrient limitation among the study lakes, followed by P limitation. Limitation type shifted with N form in 40% of the study lakes, but without consistent patterns explaining how shifts occurred. Regardless of N form, lakes with watersheds more dominated by agriculture and higher total dissolved nitrogen (TDN) tended to show P‐limited phytoplankton responses, while lakes with less agricultural watersheds and lower TDN tended to show colimited phytoplankton responses. Finally, ambient TDN and total phosphorus (TP) nutrient concentrations were stronger predictors of limitation type than ambient TDN : TP ratios. The different contributions of DON and DIN to phytoplankton biomass in some of our study lakes suggest that DON loading from surrounding watersheds may be an overlooked component in predicting phytoplankton productivity and nutrient limitation dynamics in freshwater ecosystems.