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Picophytoplankton (PP) is a dominant component of open ocean phytoplankton communities. Although this fraction may outcompete larger species in low nutrient, low light environments, its relative contribution is thought to decrease in eutrophic waters, focusing most PP research on oligotrophic systems. Recent studies suggest that PP may also be an important component of eutrophic estuarine phytoplankton assemblages. In the Neuse River Estuary (NRE) PP achieve significant biomass during certain seasons and conditions. In this study, PP biomass and primary productivity were determined to assess temporal and spatial variation of PP in the NRE. PP averaged 35.3-40.6% and 41.8-56.9% of the total biomass and primary productivity, respectively. Temperature, salinity, and nutrient concentrations were important in controlling PP dynamics; however, additional factors including grazing and estuarine residence time are likely influential. Little is known regarding the fate of PP carbon in estuaries, thus PP should not be overlooked when evaluating estuarine ecosystems.