A number of important pathogens which are resistant to penicillins and cephalosporins owe their resistance to the production of a β-lactamase. If this β-lactamase could be inhibited adequately in vivo it would be expected that infections caused by such organisms could be successfully treated with the penicillins and cephalosporins now already available. We now wish to report the isolation of certain naturally-occurring substances which are potent β-lactamase inhibitors and which also possess antibacterial activity. These substances, designated MM 14151, MM 4550 and MM 13902, are produced by certain cultures of Streptomyces. MM 14151 is produced by Streptomyces clavuligerus ATCC 27064 and has been given the trivial name clavulanic acid, which is a novel fused β-lactam structurally distinct from the penicillins, cephalosporins and cephamycins. Clavulanic acid shows a broad antibacterial spectrum but the level of activity is relatively low, however, it is a potent progressive inhibitor of the β-lactamase produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae, and in the presence of low concentrations (<10 μg/ml) of clavulanic acid many of these β-lactamase-producing organisms are rendered almost as sensitive to penicillins and cephalosporins now commercially available as are non-β-lactamase-producing strains. MM 4550 and MM 13902 are co-produced by strains of Streptomyces olivaceus (e.g. ATCC 21379) and can be distinguished chromatographically and spectroscopically from clavulanic acid and the naturally-occurring penicillins, cephalosporins and cephamycins. MM 4550, which is a sulphur-containing carboxylic acid, shows a broad-spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria, most of the sensitive organisms being inhibited at concentrations of 5-50 μg/ml, and it is also an extremely potent inhibitor of a number of β-lactamases including those produced by strains of Staph. aureus, E. coli, Klebsiella, Citrobacter, Proteus and Pseudomonas, and in the presence of MM 4550 at a concentration of 10 µg/ml or less many of these β-lactamase-producing organisms are inhibited by ampicillin at a concentration of less than 5 pg/ml. MM 13902 is also a sulphur-containing carboxylic acid related to MM 4550, it is a potent inhibitor of β-lactamase but shows considerably greater antibacterial activity and inhibits a wide range of Gram-positive and Gram-negative bacteria, including penicillin- and cephalosporin-resistant strains, with most of these organisms being inhibited by concentrations less than 5 pg/ml. Clavulanic acid is the first naturally-occurring β-lactamase inhibitor to be fully characterized and the properties of this substance and also those of MM 4550 and MM 13902 are being evaluated.