Although marine chemists can accurately quantify both the concentration of dissolved iron (Fe) and the high-affinity organic ligands which complex Fe in surface waters, tools to characterize the relative bioavailability of such organically bound Fe complexes remain unavailable. In this study, we compared the bioavailability of Fe released from the lysis of the heterotrophic bacterium Vibrio natriegens PWH3a to that of Fe complexed to synthetic chelators (EDTA) and siderophores (including the trihydroxamate desferrioxamine B [DFB] and 2 catecholates isolated from Fe-limited heterotrophic bacterial cultures) using a heterotrophic bioluminescent reporter of Fe availability (Pseudomonas putida FeLux). Using the bioluminescent response of R putida FeLux, we were able to rank the Fe sources tested here in a decreasing order of bioavailability: lysates > Fe-homologous catecholate (from a P. putida FeLux culture) similar to Fe-exogenous catecholate (from V natriegens culture) > inorganic Fe (FeCl(3), 15 nM) - Fe(III)' from EDTA-buffered treatment (pFe 18.12) > Fe:DFB, where pFe is -log [Fe(3+)]. Combined with estimates of Fe assimilation of (55)Fe-labeled lysates, our data further demonstrate that organic Fe complexes released during virus-mediated cell lysis are ca. 1000 times more bioavailable and efficiently assimilated by bacterial cells than Fe(III)'. Our results validate the utilization of P. putida FeLux as a bioreporter of Fe-bioavailability and also support the assumption that virus activity plays a crucial role in the regeneration of biologically available Fe complexes in surface seawater.