High-level expression of soluble recombinant human hemoglobin (rHb) in Escherichia coli was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108-->Lys) in beta-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type beta-globin subunit accumulated. The beta-globin Providence(asp) mutation (Lys-82-->Asp) significantly improved soluble rHb accumulation compared to the wild-type beta-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providenceasp substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-alpha-globin and beta-Lys-82-->Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% +/- 3% of the soluble cell protein in E. coli. The average total accumulation (soluble globin plus insoluble globin) was 56% +/- 7% of the soluble cell protein. Fermentations yielded 6.0 +/- 0. 3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 +/- 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in E. coli depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.