Aztreonam was investigated as to its characteristics as a substrate, inhibitor and inducer for the well-defined β-lactamases of Gram-negative bacteria, and its antibacterial efficacy as to bacterial cells producing eight types of β-lactamases was also evaluated. Aztreonam was hydrolyzed at measurable rates by class A β-lactamases, a TEM-2 type penicillinase and the Proteus vulgaris cephalosporinase with a broad substrate range. However, the affinity of aztreonam for the class A enzymes was low, this property being well reflected by its high antibacterial activity toward producers of class A β-lactamases. Aztreonam was extremely stable as to the typical class C cephalosporinase of Citrobacter freundii, and acted as a competitive and progressive inhibitor for the β-lactamase. While the MICs of aztreonam in the cases of the constitutive producers of class C β-lactamases were evidently affected by enzyme production. An experiment involving aztreonam as a inhibitor in combination with a hydrolyzable β-lactam gave ambiguous results, however, a strong synergistic effect was found in combination with mecillinam. Using Pseudomonas aeruginosa, aztreonam was confirmed to be a poor inducer of β-lactamases.