The commercial feasibility of recombinant human Hb (rHb) as an O2 delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production ofrHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28-37° C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. aGlyl5 to Ala and P Glyl6 to Ala mutations have been constructed to increase the stability of the A helices of both subunits of adult human hemoglobin (HbA), based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Human fetal hemoglobin is also known to be more stable than HbA, and comparisons between human P and y (fetal Hb) chains indicate several substitutions that stabilize the aiPi interface, one of which, pHisllb to lie, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport may enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.