<jats:p> A novel β-lactamase inhibitor has been isolated from <jats:italic>Streptomyces clavuligerus</jats:italic> ATCC 27064 and given the name clavulanic acid. Conditions for the cultivation of the organism and detection and isolation of clavulanic acid are described. This compound resembles the nucleus of a penicillin but differs in having no acylamino side chain, having oxygen instead of sulfur, and containing a β-hydroxyethylidine substituent in the oxazolidine ring. Clavulanic acid is a potent inhibitor of many β-lactamases, including those found in <jats:italic>Escherichia coli</jats:italic> (plasmid mediated), <jats:italic>Klebsiella aerogenes, Proteus mirabilis</jats:italic> , and <jats:italic>Staphylococcus aureus</jats:italic> , the inhibition being of a progressive type. The cephalosporinase type of β-lactamase found in <jats:italic>Pseudomonas aeruginosa</jats:italic> and <jats:italic>Enterobacter cloacae</jats:italic> P99 and the chromosomally mediated β-lactamase of <jats:italic>E. coli</jats:italic> are less well inhibited. The minimum inhibitory concentrations of ampicillin and cephaloridine against β-lactamase-producing, penicillin-resistant strains of <jats:italic>S. aureus, K. aerogenes, P. mirabilis</jats:italic> , and <jats:italic>E. coli</jats:italic> have been shown to be considerably reduced by the addition of low concentrations of clavulanic acid.