Spo0A-phosphate is essential for the initiation of cellular differentiation and developmental processes in Gram-positive spore-forming bacteria. Here we combined comparative genomics with analyses of microarray expression profiles to identify the Spo0A-phosphate regulon in Bacillus subtilis. The consensus Spo0A-phosphate DNA-binding motif identified from the training set based on different computational algorithms is an 8 bp sequence, TTGTCGAA. The same motif was identified by aligning the upstream regulatory sequences of spo0A-dependent genes obtained from the expression profile of Sad67 (a constitutively active form of Spo0A) and their orthologs. After the transcription units (TUs) having putative Spo0A-phosphate binding sites were obtained, conservation of regulons among the genomes of B.subtilis, Bacillus halodurans and Bacillus anthracis, and expression profiles were employed to identify the most confident predictions. Besides genes already known to be directly under the control of Spo0A-phosphate, 276 novel members (organized in 109 TUs) of the Spo0A-phosphate regulon in B.subtilis are predicted in this study. The sensitivity and specificity of our predictions are estimated based on known sites and combinations of different types of evidence. Further characterization of the novel candidates will provide information towards understanding the role of Spo0A-phosphate in the sporulation process, as well as the entire genetic network governing cellular differentiation and developmental processes in B.subtilis.