Phytoplankton interactions with iron (Fe) were examined in surface waters of Lake Erie during summer thermal stratification. Lake-wide sampling in June and September 2005 was conducted using a continuous surface water sampler (1 m sampling depth) and in July at 18 hydrographic stations (5 m sampling depth). In situ measurements of photosynthetic efficiency (maximum quantum yield of photosystem II) and phytoplankton community composition were measured using fast repetition rate fluorometry and a phytoplankton pigment-specific fluorometer, respectively, during June and September. High ratios (73%–85%) of intracellular Fe to particulate Fe coincident with increases in chlorophyll a (Chl a) concentrations in the western and central basins in June and July imply that the majority of Fe in these regions was associated with intracellular pools. Correlations between intracellular Fe and Chl a were frequently observed when Heterokontophyta and Pyrrophyta dominated the phytoplankton community. Assimilation of Fe by the phytoplankton strongly influenced its partitioning between the dissolved and particulate phase. Dissolved iron (<0.45 μm) concentrations were proportional to Chl a concentrations and both dissolved iron and Chl a were inversely proportional to nitrate concentrations in July and September, suggesting that dissolved iron influenced both nitrate drawdown and Chl a concentrations in Lake Erie surface waters in summer.