Transcription of the ctc gene in Bacillus subtilis is activated only after exponentially growing cells enter stationary phase. The promoter of the ctc gene is utilized in vitro by two minor forms of RNA polymerase, E sigma 37 and E sigma 32, but not by the most abundant form of RNA polymerase, E sigma 55. We have used the ctc promoter to direct transcription of the xylE gene on plasmid pLC4 and observed that xylE was expressed only in stationary-phase B. subtilis. We also have constructed a series of homologous plasmids that differ only by specific base substitutions in the ctc promoter. We observed that the base substitutions that affected utilization of the ctc promoter in vivo (xylE expression) were the same as those that we had previously shown to affect utilization of the promoter in vitro by E sigma 37 and E sigma 32. We conclude that it is likely that the ctc promoter is utilized in vivo by E sigma 37 or E sigma 32.