We show that the Drosophila gene diaphanous is required for cytokinesis. Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline. Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer. Null alleles are lethal. Death occurs at the onset of pupation due to the absence of imaginal discs. Mitotic figures in larval neuroblasts were found to be polyploid, apparently due to a defect in cytokinesis. The predicted 123 × 10(3) M(r) protein contains two domains shared by the formin proteins, encoded by the limb deformity gene in the mouse. These formin homology domains, which we have termed FH1 and FH2, are also found in Bni1p, the product of a Saccharomyces cerevisiae gene required for normal cytokinesis in diploid yeast cells.