Abstract Stage 0 sporulation ( spoO) mutants of Bacillus subtilis are defective in the signal transduction system initiating sporulation. Two of the products of these genes, SpoOA and SpoOF, are related to response regulator components of two-component regulatory systems used to control environmental responses in bacteria. The SpoOF response regulator was found to be the primary substrate for phosphorylation by the sporulation-specific protein kinase, Kink Phosphorylated SpoOF was the phosphodonor for a phosphotransferase, SpoOB, which transferred the phosphate group to the second response regulator, the transcription regulatory protein SpoOA. This phosphorelay provides a mechanism for signal gathering from several protein kinases using SpoOF as a secondary messenger. These divergent signals are integrated through SpoOB phosphotransferase to activate the SpoOA transcription factor. This system provides for many levels of control to prevent capricious induction of sporulation.