The phosphorelay is the signal-transduction system recognizing and integrating environmental signals to initiate sporulation. The major signal input to the phosphorelay is an ATP-dependent kinase, KinA, responsible for phosphorylating the SpoOF protein. Mutants lacking KinA, however, still sporulate, suggesting that other kinases can fulfil its role. In order to identify these kinases, genes for kinases were isolated by hybridization using a degenerate oligonucleotide probe designed for common regions of this class of kinases. A gene for a second kinase, KinB, was isolated which gave a sporulation negative phenotype when inactivated in a kinA background. The kinB locus was sequenced and found to be a small operon consisting of the kinB gene and another gene, kapB, transcribed from a single.sigma A.-dependent promoter. Inactivation of either kinB or kapB in a kinA strain led to severe sporulation deficiency. The kinB gene coded for a 47774 M(r) protein with the carboxyl half of this protein highly homologous to the same domain of KinA. The amino-terminal domain of KinB was hydrophobic with six recognizable membrane-spanning regions. The kapB gene coded for a moderately charged, probably soluble, protein of 14,668 M(r) with no homology to any known protein. Genetic evidence suggests that KapB is required either for the function of KinB or for its expression. Although double mutants kinA kinB cannot sporulate and assume a stage 0 phenotype, the SpoA approximately P-dependent regulation of the abrB gene is normal in these strains, suggesting that low levels of SpoA approximately P accumulate even in the absence of both kinases. This accumulation is dependent on functional spo0F and spo0B genes and its source is unknown. The KinA and KinB pathways are the only pathways capable of producing sufficient Spo0A approximately P to allow initiation and completion of sporulation under laboratory conditions.