Tropomyosin (TM) is an actin binding protein that has been implicated in a variety of developmental and biological processes including neuronal morphogenesis, cell transformation, and cell motility (Gunning et al., 2008; Michelot and Drubin, 2011). The precise roles of TM in these processes has remained murky, largely because of the great diversity of TM isoforms; human cells for example express up to forty TM isoforms from four genes, most of which are poorly characterized (Gunning et al., 2005). In Drosophila there are two TM genes (Tm1 and Tm2) to which various functions have been attributed, but splicing complexity and nomenclature changes have inhibited identifying specific TM isoforms and their cellular roles (Bautch and Storti, 1983; Erdélyi et al., 1995). Here, we have carefully identified and characterized three nonmuscle TM isoforms expressed in Drosophila S2 cells. We found that all three TM isoforms have both overlapping and distinct functions that are cell cycle specific, including localizations and functions at the lamellum, Golgi, and cortex during interphase, and the cortex, kinetochores, and spindle apparatus during mitosis. Using GFP-tagged TM chimeras, we have also identified regions of individual TM isoforms that are responsible for the localization and functional differences. Together, these experiments have led to functional insights that, combined with previously known functions of tropomyosin isoforms, allow us to propose a model of how different nonmuscle TM isoforms function together in S2 cells throughout the cell cycle. Our results implicate Drosophila TM in cell cycle control, chromosome segregation, cellular contractility, and Golgi morphogenesis.