ABSTRACT The ability to remove a genetic function from an organism with good temporal resolution is crucial for characterizing essential genes or genes that act in complex developmental programs. The rhizobium-legume symbiosis involves an elaborate two-organism interaction requiring multiple levels of signal exchange. As an important step toward probing rhizobium genetic functions with temporal resolution, we present the development of a conditional gene deletion system in Sinorhizobium meliloti that employs Cre/ loxP site-specific recombination. This system enables chemically inducible and irreversible gene deletion or gene upregulation. Recombinase-mediated excision events can be positively or negatively selected or monitored by a colorimetric assay. The system may be adaptable to various bacterial species, in which recombinase activity may be placed under the control of diverse user-defined promoters. This system also shows promise for uses in promoter trapping and biosensing applications.