Spo0A is both a positive and a negative transcriptional regulator which plays a very important role in sporulation initiation in Bacillus subtilis. Its N-terminal amino acid sequence is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria. Phosphorylation of Spo0A through phosphorelay has been reported by Burbulys et al. (1991). In this study, we found that (i) Spo0A is phosphorylated effectively with phospho-EnvZ* (N-terminal truncated EnvZ), which is a heterologous osmotic sensor protein in Escherichia coli, and (ii) a phosphorylation deficient mutant of Spo0A protein is completely defective in initiating sporulation. These results suggest that Spo0A phosphorylation may be an essential event in signal transduction of sporulation in B. subtilis and the signal transduction mechanism has a common feature in Gram-positive and Gram-negative bacteria.