KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall.