Proteins that catalyze H-2-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O-2. We tested whether deletion of the gene encoding the transcriptional negative regulator, IscR, could enhance the ability of Escherichia coli BL21 to synthesize active recombinant H-2-pathway components and stimulate ferredoxin-dependent H-2-accumulation in the presence or absence of oxygen. Under anoxic conditions, deletion of iscR stimulated recombinant Fe-Fe hydrogenase activity threefold, whilst plasmid-based overexpression of the isc operon had no effect on hydrogenase activity. After cultivation with 21% (v/v) O-2 in the headspace, no recombinant hydrogenase activity was observed in soluble extracts of wild-type BL21, although low levels of activity could be observed in the Delta iscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O-2, Delta iscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H-2-accumulation relative to the control strain. In cultures starting with 10% (v/v) O-2, H-2-accumulation was stimulated 35-fold relative to the control. Delta iscR strains displayed enhanced synthesis and activity of integral H-2-pathway components under all tested conditions and enhanced H-2-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H-2-production, particularly if the hydrogenase catalyzes the rate-limiting reaction.