Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress. Activation of sporulation-specific gene transcription is under the control of the spoOA gene product. The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges. Response regulators are activated by specific kinases which phosphorylate them. In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus. The phosphodonor in this reaction is the phosphorylated form of SpoOF. It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress. The series of phosphate transfer reactions in this system is called a phosphorelay. The end product of this series of reactions is SpoOA approximately P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form. SpoOA approximately P, but not SpoOA, was shown to be an activator of transcription of the spoIIA operon which codes for the sporulation-specific sigma factor sigma F. Thus, the initiation of sporulation is dependent on SpoOA approximately P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g. abrB, and activate others, e.g. spoIIA.