In cell-walled organisms, a cross wall (septum) is produced during cytokinesis, which then splits in certain organisms to allow the daughter cells to separate. The formation and the subsequent cleavage of the septum require wall synthesis and wall degradation, which need to be strictly coordinated in order to prevent cell lysis. The dividing fission yeast (Schizosaccharomyces) cell produces a three-layered septum in which the middle layer and a narrow band of the adjacent cell wall can be degraded without threatening the integrity of the separating daughter cells. This spatially very precise process requires the activity of the Agn1p 1,3-alpha-glucanase and the Eng1p 1,3-beta-glucanase, which are localized to the septum by a complex mechanism involving the formation of a septin ring and the directed activity of the exocyst system. The Sep1p-Ace2p transcription-factor cascade regulates the expression of many genes producing proteins for this complex process. Recent advances in research into the molecular mechanisms of separation and its regulation are discussed in this review.