Nucleotides were effective in inhibiting the class C beta-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a K-i value of 16.2 mu M. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.