The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology. The cryptic nature of expression of leuO has previously limited such analysis. A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3',5'-bispyrophosphate-dependent. Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3',5'-bispyrophosphate. In this study, we tested whether LeuO was directly involved in the bacterial stringent response. LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E. coli K-12 relA1 strain. This result supports a functional role for LeuO in the bacterial stringent response.