Although many proteins can be overexpressed several fold without much effect on cell viability and morphology, some become toxic upon a slight increase in their intracellular level. This is particularly true for cytoskeletal proteins and has proven useful in the past for studying the cytoskeleton. In yeast, actin and tubulin are examples of proteins that cannot be overexpressed without affecting cell viability. Here, we have analysed the effect of actin overexpression in Saccharomyces cerevisiae. We show that actin overexpression interferes differently with distinct aspects of actin function. For example, two- to fourfold overexpression of actin did not affect the establishment of actin polarity, whereas it abrogated its maintenance. Also, actin structures that are barely visible in wild-type cells could be observed upon actin overexpression. This allowed us to identify a new ring-like actin structure genetically distinguishable from the actomyosin contractile ring. Formation of this actin structure upon actin overexpression was dependent on the septin cytoskeleton, the poorly understood cytokinetic protein Hof1 and the Arp2/3 complex. In contrast to the actomyosin ring, the ring formed upon actin overexpression required neither Myo1 nor formins for assembly. Therefore, we propose that Hof1 acts as a linker between actin and septins. Furthermore, we found that, in the absence of actin overexpression, a novel, Hof1-dependent actin belt is formed at the bud neck of anaphase cells. The physiological role of this belt might be related to that of the similar structure observed in dividing fission yeast.