Studies of cytokinesis in animal cells demonstrate that microtubules play an important role in signaling the position of the actin-containing contractile ring and subsequent formation of the cleavage furrow. Septation in several fungi closely resembles animal cell cytokinesis in that a circumferential ring of actin is visible at the incipient division site. However, this does not necessarily mean that division is contractile since actin may also serve to localize septal wall synthesis. In addition, several studies in fission yeast have suggested that microtubules are dispensable for actin ring formation. We have used synchronized cells and fluorescence microscopy to follow actin structures, nuclear division and septal wall synthesis during septation in Aspergillus nidulans. Our data suggest that actin first appears at the septum site as a circumferential ring and that it later broadens and invaginates, forming an hourglass-shaped structure coincident with septal cell wall synthesis. Depolymerization of microtubules early in septation prevents circumferential actin ring formation. Depolymerization of microtubules after circumferential actin ring formation blocks both the progression to invaginating bands and septal wall synthesis. In contrast to studies in yeast cells, our data suggest that microtubules are required for both the initiation and progression of septation in A. nidulans.