Significance and Impact of the Study: The enzyme urease is a virulence factor for the Gram-positive urinary tract pathogen Staphylococcus saprophyticus. We have shown that urease activity in cell-free extracts and whole bacterial cells is susceptible to inhibition by hydroxamates, phosphorodiamidates and flavonoids, but not by imidazoles. Acetohydroxamic acid and fluorofamide in particular can temporarily delay the increase in pH that occurs when Staph. saprophyticus is grown in an artificial urine medium. These results suggest that urease inhibitors may be useful as chemotherapeutic agents for the treatment of urinary tract infections caused by this micro-organism. AbstractUrease is a virulence factor for the Gram-positive urinary tract pathogen Staphylococcus saprophyticus. The susceptibility of this enzyme to chemical inhibition was determined using soluble extracts of Staph. saprophyticus strain ATCC 15305. Acetohydroxamic acid (K i = 8Á2 lg ml À1 = 0Á106 mmol l À1 ) and DL-phenylalanine hydroxamic acid (K i = 21 lg ml À1 = 0Á116 mmol l À1 ) inhibited urease activity competitively. The phosphorodiamidate fluorofamide also caused competitive inhibition (K i = 0Á12 lg ml À1 = 0Á553 lmol l À1 = 0Á000553 mmol l À1 ), but the imidazole omeprazole had no effect. Two flavonoids found in green tea extract [(+)-catechin hydrate (K i = 357 lg ml À1 = 1Á23 mmol l À1 ) and (À)-epigallocatechin gallate (K i = 210 lg ml À1 = 0Á460 mmol l À1 )] gave mixed inhibition. Acetohydroxamic acid, DL-phenylalanine hydroxamic acid, fluorofamide, (+)-catechin hydrate and (À)-epigallocatechin gallate also inhibited urease activity in whole cells of strains ATCC 15305, ATCC 35552 and ATCC 49907 grown in a rich medium or an artificial urine medium. Addition of acetohydroxamic acid or fluorofamide to cultures of Staph. saprophyticus in an artificial urine medium delayed the increase in pH that normally occurs during growth. These results suggest that urease inhibitors may be useful for treating urinary tract infections caused by Staph. saprophyticus.