Updated project metadata. In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by Histidine or Serine/Threonine kinases are key points in this regulation. PrkA has been proposed to be an essential Serine kinase for the initiation of sporulation but its kinase activity has not been clearly demonstrated so far. Indeed, neither its autophosphorylation nor identification of a B. subtilis phosphorylated substrate was unambiguously established. Bioinformatic homology searches revealed sequence similarities with the AAA+ ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyse the α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr kinases of B. subtilis, PrkC, as characterized by mass spectrometry from 3 in vitro independent experiments (1: PrkA+PrkC/2:PrKA-S219E+PrkC/3: PrKA-T217E+PrkC). We finally demonstrated that PrkA regulation of the transcriptional factor σK via the transition phase regulator ScoC is certainly indirect.