The initiation of the sporulation developmental pathway in Bacillus subtilis is controlled by the phospho-relay, a multicomponent signal transduction system. Multiple positive and negative signals are integrated by the phosphorelay through the opposing activities of histidine protein kinases and aspartyl phosphate phosphatases. Three members of the Rap family of phosphatases (RapA, RapB and RapE) specifically dephosphorylate the Spo0F approximately P response regulator intermediate, while the Spo0A approximately P transcription factor is specifically dephosphorylated by the Spo0E phosphatase and, as shown here, the newly identified YnzD and YisI proteins. The products of the YnzD and YisI genes are highly homologous to Spo0E and define a new family of phosphatases with a distinct signature motif in their amino acid sequence. As negative regulators of the developmental pathway, YnzD and YisI inhibit spore formation if over-expressed, while a chromosomal deletion of their coding sequences results in increased sporulation frequency. Transcription of the ynzD, yisI and spo0E genes is differentially regulated and generally induced by growth conditions antithetical to sporulation. Negative signals interpreted by aspartyl phosphate phosphatases appear to be a common mechanism in Gram-positive spore-forming microorganisms.