To better understand Fe cycling in marine and freshwater systems, we have developed a biomolecular tool to track the perceived bioavailability of Fe to heterotrophic bacteria. Bioluminescent reporters, constructed by fusing the fepA–fes promoter of Escherichia coli (an Enterobactin biosynthesis gene regulated by the ferric uptake regulatory [Fur] system) to a luxCDABE cassette, were integrated into the chromosome of a halotolerant Pseudomonas putida, which uses the Fur system to regulate high-affinity Fe uptake. The resultant P. putida bioreporter has been successfully tested both in lab and field studies. Laboratory cultures were maintained at a range of concentrations of total Fe (0–25 nM) or limited by the addition of concentrations of well-characterized siderophores (desferrioxamine B [DFB], ferrichrome, 2,2′-dipyridyl [DP] and Rhodotorulic acid [RA], 0–200 nM) and used to establish the dynamic range of this reporter system. Analysis of sample incubations after only 4 h suggest that both of the trihydroxamate-type siderophores DFB and ferrichrome efficiently reduced Fe availability, resulting respectively in a 1.77- and 1.88-fold increase in luminescence relative to Fe-replete conditions. In contrast, additions of the dihydroxamate-type siderophore RA and the synthetic chelator DP resulted in no response from the system, suggesting that cells could access Fe complexed to these compounds without activating high-affinity Fe transport systems.Field studies were performed in the central basin of Lake Erie, which has previously been shown to undergo sporadic Fe limitation during summer stratification. DFB concentrations were titrated across a range of 0–50 nM into unfiltered water to manipulate Fe availabilities. Bioreporters expressed Fe stress (ca. a 2-fold increase in luminescence) at concentrations of DFB equivalent to the total (dissolved+particulate) Fe in the system (≈30 nM), indicative of the concentration of bioavailable Fe. In a similar experiment with 0.2-μm pre-filtered water (2.25–5.24 nM Fe), a 6-fold increase in luminescence (relative to controls) was observed at the lowest (15 nM) concentration of chelators. The results of this study demonstrate the validity of bioreporters as a complimentary tool to measurements of total Fe. Moreover, these results suggest that a significant source of bioavailable Fe flux may be particulate in nature, and support the hypothesis that Fe recycling from microorganisms may play a key role in the regeneration of bioavailable forms of Fe.