Supply of anthropogenic nitrogen (N) to the biosphere has tripled since 1960; however, little is known of how in situ response to N fertilisation differs among phytoplankton, whether species response varies with the chemical form of N, or how interpretation of N effects is influenced by the method of analysis (microscopy, pigment biomarkers). To address these issues, we conducted two 21-day in situ mesocosm (3140 L) experiments to quantify the species- and genus-specific responses of phytoplankton to fertilisation of P-rich lake waters with ammonium (NH 4+), nitrate (NO 32), and urea ([NH 2 ]2 CO). Phytoplankton abundance was estimated using both microscopic enumeration of cell densities and high performance liquid chromatographic (HPLC) analysis of algal pigments. We found that total algal biomass increased 200% and 350% following fertilisation with NO 32 and chemically-reduced N (NH 4+, urea), respectively, although 144 individual taxa exhibited distinctive responses to N, including compound-specific stimulation (Planktothrix agardhii and NH 4+), increased biomass with chemically-reduced N alone (Scenedesmus spp., Coelastrum astroideum) and no response (Aphanizomenon flos-aquae, Ceratium hirundinella). Principle components analyses (PCA) captured 53.2–69.9% of variation in experimental assemblages irrespective of the degree of taxonomic resolution of analysis. PCA of species-level data revealed that congeneric taxa exhibited common responses to fertilisation regimes (e.g., Microcystis aeruginosa, M. flos-aquae, M. botrys), whereas genera within the same division had widely divergent responses to added N (e.g., Anabaena, Planktothrix, Microcystis). Least-squares regression analysis demonstrated that changes in phytoplankton biomass determined by microscopy were correlated significantly (p,0.005) with variations in HPLC-derived concentrations of biomarker pigments (r2 = 0.13–0.64) from all major algal groups, although HPLC tended to underestimate the relative abundance of cyanobacteria. Together, these findings show that while fertilisation of P-rich lakes with N can increase algal biomass, there is substantial variation in responses of genera and divisions to specific chemical forms of added N.