Abstract: An understanding of cytokinesis at the molecular level requires a detailed description of the protein complexes that perform central activities during this process. The proteins Hof1p, Cyk3p, Inn1p and Myo1p each represent one of the four genetically defined and partially complementary pathways of cytokinesis in the yeast Saccharomyces cerevisiae. Here we show that the osmosensor Sho1p is required for correct cell-cell separation. Shortly before cytokinesis Sho1p sequentially assembles with Hof1p, Inn1p and Cyk3p, into a complex (the HICS complex) that might help to connect the membrane with the actin-myosin ring. The HICS complex is formed exclusively through interactions between three SH3 domains located in Cyk3p, Hof1p and Sho1p, and five acceptor sites found in Cyk3p, Hof1p and Inn1p. Owing to the overlapping binding specificities of its members the HICS complex is best described as ensembles of isomeric interaction states that precisely coordinate the different functions of the interactors during cytokinesis.