The genomic locus responsible for production of the globin portion of Vitreoscilla hemoglobin (VtHb), the only well-characterized bacterial hemoglobin (Hb), has been cloned and expressed in Escherichia coli. A 17-mer oligodeoxynucleotide, corresponding to a region of the VtHb amino acid sequence was used as a hybridization probe to screen a Vitreoscilla genomic library constructed in broad-host-range cosmid vector pVK102. E. coli, carrying recombinant pVK102: H5 which contained a 16.5-kb insert of Vitreoscilla genomic DNA, produced three to four times more Hb than Vitreoscilla. Restriction mapping and subcloning revealed that the globin-coding gene (vgb) was completely localized on a 1.4-kb HindIII-SalI fragment of the 16.5-kb insert. Production of VtHb still occurred when this 1.4-kb fragment was cloned in plasmids pUC8 and pUC9 in opposite orientations, suggesting the presence of a Vitreoscilla promoter on this fragment. A single copy of this gene on the chromosome was indicated by Southern-blot analysis, and a 450–500-nt RNA transcript specific for the globin gene was detected after Northern hybridization. A partially purified Hb preparation from E. coli harboring the recombinant plasmid had identical spectral properties and subunit molecular size as authentic VtHb. The Hb in respiring cells of E. coli was in the physiologically functional oxyHb form.