Spore formation by Bacillus subtilis is governed by global changes in gene transcription. We used nylon-substrate DNA arrays representing Ϸ96% of the predicted open reading frames in the B. subtilis chromosome to compare the pattern of transcripts from wild-type cells with the pattern from cells mutant for the sporulation transcription factors Spo0A or F . We found 520 genes whose transcript levels were at least 3-fold dependent on Spo0A but not on F , and an additional 66 genes whose transcript levels were dependent upon both regulatory proteins. Two strategies were used to help assign genes to the direct control of a particular developmental regulatory protein. In one approach, we analyzed the effects on global gene expression of artificially producing a constitutively active form of Spo0A during growth. In a second approach, Hidden Markov models were used to identify promoters likely to be activated by Spo0A, F , or a third sporulation transcription factor, E . In addition to detecting known sporulation genes, we identified many genes of unknown function whose patterns of expression and regulation suggest that they could be involved in sporulation. Disruption of two such newly identified genes, yabP and yabQ, blocked sporulation at a late stage.hidden Markov models ͉ functional genomics ͉ DNA arrays S porulation by Bacillus subtilis is governed by a program of gene transcription involving more than 125 genes (reviewed in ref. 1). However, a description of global changes in gene expression during the course of spore formation has been lacking. Recent methods for creating DNA arrays with large numbers of individual genes has made it possible to visualize genome-wide changes in gene transcript levels, as, for example, during sporulation in budding yeast and during the response of human fibroblasts to serum (2, 3). Taking advantage of the availability of a complete genome sequence for B. subtilis (4), we sought to apply transcriptional profiling to the process of sporulation in this bacterium.Entry into sporulation is principally governed by the DNAbinding protein Spo0A, which is both an activator and a repressor of transcription. The activity of Spo0A, which is a member of the response regulator family of proteins, is controlled through phosphorylation by a phosphorelay that integrates environmental and physiological signals in the decision to sporulate (5). Among the targets of the phosphorylated form, Spo0AϳP, are one or more unknown genes involved in the formation of a polar septum, which divides the cell into forespore and the mother cell compartments (6). Spo0AϳP also directs the transcription of genes and operons (spoIIA, spoIIE, spoIIG) involved in the activation of the compartment-specific regulatory proteins Two additional approaches were used to identify genes under the control of Spo0A and other regulatory proteins. In one, we monitored gene expression in cells engineered to produce a constitutively active form of Spo0A (Spo0A-Sad67) during growth (9). In a second approach, we used hidden Markov mode...