Iron enrichments in bottle experiments in high nutrient, low chlorophyll (HNLC) surface waters typically stimulate the net growth of nanophytoplankton (2.0–20 µm) but not picophytoplankton (<2.0 µm), suggesting either that picophytoplankton are not Fe limited or that any increased picophytoplankton growth is balanced by increased microzooplankton grazing rates. Using a novel approach in which we diminish iron availability in seawater with the fungal siderophore deferriferrioxamine B, we demonstrate that growth of the abundant picoplanktonic cyanobacterium Synechococcus is not strongly rate limited by Fe in the HNLC equatorial Pacific Ocean. However, experiments at the equator and 5°S show an unsuspected non-uniformity in iron nutrition within this HNLC region. Furthermore, our results indicate that a major portion of “dissolved” (<0.4 µm) iron in these waters is unavailable to Synechococcus, providing the first evidence that a significant fraction of dissolved iron occurs in forms other than the simple hydroxy species predicted by thermodynamic models. Deferriferrioxamine B affords a powerful new tool for probing the iron nutrition of marine phytoplankton and can provide unique insight into the role of iron in marine biogeochemical cycles.