The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3.5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.