The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay, a complex signal transduction system consisting of kinases and response regulators. The key component of a phosphorelay is the phosphotransferase, which recognizes two response regulators and transfers a phosphoryl group between them. In this reaction, the phosphoryl of one response regulator is transferred to a histidine on the phosphotransferase before phosphorylating an aspartate of the second response regulator. The phosphorylated histidine on the Spo0B phosphotransferase was found to be His30. Site-directed mutation of His30 to alanine destroyed its phosphotransferase activity in vitro and strains constructed with this mutation were unable to sporulate. None of the other 10 histidines of Spo0B was implicated in the phosphotransferase reaction. A structurally vulnerable site, histidine 23, was also identified through the mutational study. The His30 of Spo0B resides in a domain with little sequence homology to functionally equivalent domains in the phosphorelays of other bacterial and yeast systems, suggesting that the two types of phosphotransfer domains evolved convergently.