This study describes a short-term (12 h) evaluation of iron (Fe) bioavailability to an Fe-dependent cyanobacterial bioreporter derived from Synechococcus PCC 7942. Several synthetic ligands with variable conditional stability constants for Fe(III) (K* of 1019.8 to 1030.9), in addition to several defined natural Fe-binding ligands and a fulvic acid of aquatic origin (Suwannee River), were used to elucidate the forms of Fe that are discerned by this phytoplanktonic microbe: Fe-HEBD (log conditional stability constant, K*= 28.1, HEBD=N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid monohydrochloride hydrate), Fe-HDFB(K*= 30.9, DFB=desferroxamine B), Fe-ferrichrome(K*= 23.2), Fe-DTPA (K*= 21.1, DTPA= diethylenetrinitrilopentaacetic acid), Fe-(8HQS)2 (K*= 20.4, 8HQS= 8-hydroxyquinoline-5-sulfonic acid), Fe-CDTA (K*= 19.8, CDTA= trans-1,2-cyclohexylenedinitrilotetraacetic acid), and Fe-EDTA (K*= 19.2). Iron bioavailability sensed by the bioreporter was related to diffusion limitation and activity of high-affinity transporters rather than by siderophore secretion. Iron complexed with a K* < 23.2 contributes to the bioavailable pool; bioavailability could be explained by disjunctive ligand exchange considerations and fully, partially, and nonbioavailable complexes could be distinguished according to their conditional stability constant. The use of Fe-bioreporters provides a relevant measurement of bioavailability to an important group of primary producers in freshwaters (cyanobacteria) and is thus a promising technique for understanding Fe cycling in aquatic systems.