Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Only one nonmuscle tropomyosin (Tm1A) has previously been described in Drosophila, but developmental defects caused by insertion of P-elements near tropomyosin genes imply the existence of additional, nonmuscle isoforms. Using biochemical and molecular genetic approaches, we identified three tropomyosins expressed in Drosophila S2 cells: Tm1A, Tm1J, and Tm2A. The Tm1A isoform localizes to the cell cortex, lamellar actin networks, and the cleavage furrow of dividing cells--always together with myosin-II. Isoforms Tm1J and Tm2A colocalize around the Golgi apparatus with the formin-family protein Diaphanous, and loss of either isoform perturbs cell cycle progression. During mitosis, Tm1J localizes to the mitotic spindle, where it promotes chromosome segregation. Using chimeras, we identified the determinants of tropomyosin localization near the C-terminus. This work 1) identifies and characterizes previously unknown nonmuscle tropomyosins in Drosophila, 2) reveals a function for tropomyosin in the mitotic spindle, and 3) uncovers sequence elements that specify isoform-specific localizations and functions of tropomyosin.