Iron bioavailability in Lake Superior was assessed during field surveys conducted in 2001-2002. Dissolved iron (Fed) ranged between 1 and 4 nM at offshore stations and >10 nM at most nearshore sites. Iron availability was assessed using a luminescent Synechococcus bioreporter comprising a luciferase reporter controlled by an iron-responsive promoter isiAB. Bioreporter luminescence was negatively correlated to Fed measured in the samples. Distance from shore was a better predictor of iron bioavailability than was season. Water collected from most offshore stations sampled during spring and summer elicited higher bioreporter luminescence than did nearshore sites. Iron availability did not vary with depth during summer, despite the presence of elevated levels of Fed in the hypolimnion at most stations. Ultrafiltration (0.02 μm) of Fed at two offshore sites demonstrated Fed to be present mainly in a colloidal phase, yet the bioreporter responded solely to iron contained in the soluble phase. During spring, a parallel immunochemical assay of diatoms resulted in the detection of ferredoxin (Fd) but not flavodoxin (Flvd) at five stations indicating the presence of an iron sufficient assemblage of diatoms at these sites. Whereas neither bioreporter nor immunochemical approaches conducted during spring supported physiological iron deficiency among Lake Superior phytoplankton, the results did point to differences in the availability of iron to prokaryotes and eukaryotes.