Mutation in the gene sep1+ of the unicellular fission yeast Schizosaccharomyces pombe impairs cell separation after cytokinesis and confers a branching mycelial morphology. The mutant is not defective in cell wall beta-glucanase activity but shows increased sensitivity to Ca2+ and Mg2+, and increased resistance to the microtubule inhibitor benomyl. The mycelial growth of sep1-1 provides a convenient method for the examination of the polar growth pattern and for pedigree analysis as demonstrated by the segregation of mating types in the homothallic microhyphae. sep1 is closely linked to ade1 (0.94 cM) on the right arm of chromosome II. The ts mutation spl1-1 confers a bent cell shape and causes aberrant septum formation at the restrictive temperature. sep1+ and spl1+ perform closely related functions as their mutant alleles interact with each other and with another septation mutant cdc4-8. These functions may overlap with certain cytoskeletal processes and with the determination of cell polarity because the triple mutant forms huge multinucleate syncytia with promiscuous branching and rare septum formation.