The methylotrophic bacterium Methylophilus methylotrophus hydrolyses urea to ammonia using a cytoplasmic urease (EC 3.5.1.5). During growth in continuous culture under various nutrient limitations, urease was induced by urea and short-chain amides (formamide and urea acetamide), and repressed by excess ammonia. The enzyme was purified using ammonium sulfate fractionation and fast protein liquid chromatography (FPLC), and exhibited a narrow substrate specificity (urea formamide and acetamide; no activity with other amides) with a K m for urea of 3.8 mM. Gel filtration FPLC and SDS-PAGE showed that the enzyme had a native M r of approximately 190 000 and was composed of α (M r 64 000), β (M r 15 500) and γ (M r 15 000) subunits in the probable ratio α 2 β 2 γ 2 . The physiological regulation and biochemical properties of the M. methylotrophus urease are compared with those of other bacterial ureases.