Beta-lactam antibiotics are among the most important drugs used to fight bacterial infection. Overuse and misuse of beta-lactam antibiotics has caused the evolution of resistance mechanisms, allowing pathogenic bacteria to survive antibiotic treatment. The major source of resistance to beta-lactam antibiotics occurs through production of enzymes called beta-lactamases capable of catalyzing hydrolysis of the beta-lactam rings in these drug compounds. The metallo-beta-lactamases have become a major threat due to their broad substrate specificities; there are no clinically useful inhibitors for these metalloenzymes. We have obtained single-stranded DNA's that are potent inhibitors of the Bacillus cereus 5/B/6 metallo-beta-lactamase. These are rapid, reversible, non-competitive inhibitors of the metalloenzyme, with K(i) and K(i)' values in the nanomolar range. The inhibition patterns and metal ion dependence of their inhibition suggest that the oligonucleotides alter the coordination of the active site metal ion(s); inhibition is efficient and highly specific. Microbiological growth experiments, using combinations of ssDNA with the beta-lactam antibiotic cephalexin, reveal that the inhibitor is capable of causing cell death in liquid cultures of both Gram-positive and Gram-negative metallo-beta-lactamase producing bacteria in the micromolar concentration range.