We examined seasonality in the phosphorus (P) physiology of the phytoplankton assemblage of culturally hypereutrophic Onondaga Lake, an urban system located in metropolitan Syracuse, NY. Four years of monitoring data for ambient dissolved and cellular phosphorus pools and phytoplankton biomass and one year of alkaline phosphatase activity (APA) assays supported the assessment. Kinetic approaches utilizing ambient dissolved and stored nutrient pools were evaluated for their utility in tracking nutrient limitation and gave evidence for luxury uptake of soluble reactive P (SRP) and utilization of the dissolved organic P (DOP) pool by the lake's phytoplankton. The high concentrations of phytoplankton biomass maintained annually from mid-to late summer were sustained in part by the DOP pool. Concentrations of SRP appear to be an inadequate representation of nutrient limitation in the lake. The cellular concentration of P, represented by the cell quota (Q, μg particulate P /μg chlorophyll), was generally lower when SRP was depleted. APA is a sensitive indicator of the utilization of DOP and the occurrence of nutrient limitation. The threshold Q value for inducement of APA in the lake is about 1 μgPPμgChl−1. The onset of APA can be used to identify conditions of resource limitation where further reductions in nutrient availability may be expected to yield proportional improvements in water quality. A kinetic framework that relates growth rate to cellular nutrient concentration (Droop kinetics) is favored over one based on ambient dissolved nutrient concentrations (Monod kinetics) for quantifying nutrient limitation in Onondaga Lake.