Streptomyces clavuligerus has been shown to produce penicillin N, 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid, 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid, and deacetoxycephalosporin C. During a programme of screening for novel β-lactamase inhibitors, a new type of β-lactam antibiotic, clavulanic acid [Z-(2R,5R)-3-(p-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid], was discovered from this organism. The structure of clavulanic acid, a β-lactamase inhibitor, was investigated using spectroscopic methods and X-ray analysis. Methylation of sodium clavulanate gave methyl clavulanate, whose n.m.r. and ¹³C n.m.r. spectra provided insights into the β-lactam system and other structural features. The gross structure was proposed as (1) or its E-isomer. Structure (1a) and relative stereochemistry were elucidated by X-ray analysis of the p-nitrobenzyl ester (1c), while the absolute configuration was established by X-ray analysis of the p-bromobenzyl ester (1d) using the heavy atom method and anomalous dispersion. Clavulanic acid is unique as the first naturally occurring fused β-lactam containing oxygen instead of sulphur and lacking the acylamino-side chain present in penicillins and cephalosporins. As its sodium salt, it is a potent irreversible inhibitor of various β-lactamases. When combined with ampicillin (5 µg/ml) in vitro, it reduced the minimum inhibitory concentration of ampicillin from >1000 µg/ml to <1.0 µg/ml against a β-lactamase-producing strain of Staphylococcus aureus.