It is conventional knowledge that heterotrophic bacteria play a key role in the biogeochemical cycling of oceanic carbon. However, only recently has their role in marine iron (Fe) biogeochemical cycles been examined. Research during this past decade has demonstrated an inextricable link between Fe chemistry and the biota, as >99% of Fe in marine systems is complexed to organic chelates of unknown but obviously biotic origin. Here we present a novel approach to assess and compare Fe bioavailability in low Fe HNLC waters using a bioluminescent bacterial reporter that quantitatively responds to the concentration of bioavailable Fe by producing light. Originally tested in freshwater environments, this study presents the first characterization of this halotolerant reporter organism in a defined seawater medium and then subsequently in marine surface waters. Laboratory characterizations demonstrate that this reporter displays a dose-dependent response to Fe availability in our defined marine medium. Field tests were performed during the 10-day mesoscale FeCycle experiment (February 2003) in the Pacific sub-Antarctic high-nitrate low-chlorophyll region. Data from both biogeochemical measures and bioreporter assays are provided which describe how the bioreporter detected changes in Fe bioavailability that occurred during a natural shift in ambient dissolved Fe concentrations (∼40 pM). Our data explore the use of heterotrophic bioluminescent reporters as a comparable tool for marine ecosystems and demonstrate the potential utility of this tool in elucidating the relationship between Fe bioavailability and Fe chemistry in complex marine systems.