A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-5'-monophosphate (c-di-GMP) have been created by using in vitro selection. An RNA library was generated by using random-sequence bridges to join a hammerhead self-cleaving ribozyme to an aptamer from a natural c-di-GMP riboswitch. Specific bridge sequences, called communication modules, emerged through two in vitro selection efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptamer. Representative RNAs were found that exhibit EC(50) (half-maximal effective concentration) values for c-di-GMP as low as 90 nM and IC(50) (half-maximal inhibitory concentration) values as low as 180 nM. The allosteric RNAs display molecular recognition characteristics that mimic the high discriminatory ability of the natural aptamer. Some engineered RNAs operate with ribozyme rate constants approaching that of the parent hammerhead ribozyme. By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in three mutant strains of Escherichia coli were quantitatively estimated from cell lysates. Our findings demonstrate that engineered c-di-GMP-sensing ribozymes can be used as convenient tools to monitor c-di-GMP levels from complex biological or chemical samples. Moreover, these ribozymes could be employed in high-throughput screens to identify compounds that trigger c-di-GMP riboswitch function.