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Ampicillin sensitivity tests and tests for ampicillin inactivation were made with 148 strains of coliform bacilli. A correlation was found in strains of Proteus mirabilis and Klebsiella between ampicillin resistance and inactivation. No correlation was obtained with Escherichia coli, whether sensitive or resistant to ampicillin, although small amounts of penicillinase were produced. Ampicillin-sensitive strains of P. mirabilis showed even lower amounts of penicillinase. The strains of Pseudomonas pyocyanea (aeruginosa) were resistant to ampicillin, but also showed low penicillinase levels. No evidence of induction of penicillinase was obtained with E. coli or Klebsiella in the presence of methicillin, ampicillin or cephalosporin C. The ampicillin-inactivating enzyme is a β-lactamase, not an amidase. Resistance of ampicillin-sensitive strains of E. coli was increased rapidly by serial subcultures but was not associated with higher penicillinase values. Ampicillin was inactivated less rapidly than was benzylpenicillin by penicillinase from an E. coli strain.