Polarized deposition of chitin at the bud neck is essential for cell separation in yeast. Chitin septum biogenesis is catalyzed by two distinct chitin synthase activities encoded by the CHS2 and CHS3 genes. The phosphoinositide phosphatase Sac1p is required for proper trafficking of the Chs3p chitin synthase. sac1 mutants also display a severe synthetic growth defect, with mutations in the SLT2 gene which encodes a MAP kinase involved in cell integrity. We characterized the defect that underlies this genetic interaction and found that sac1Delta slt2Delta cells arrest as large-budded cells because they fail to separate at the end of mitosis. This inability to complete cell division appears to be caused by an increased deposition of chitin at the septum area and correlates with a mislocalized accumulation of the Chs2p chitin synthase at the cell periphery. Our data therefore indicate that Sac1p and Slt2p have synergistic roles in regulating chitin septum biogenesis.