We have used murine splenic erythrolasts infected with the anemia-inducing strain of Friend virus (FVA cells), as an in vitro model to study cytoskeletal elements during erythroid maturation and enucleation. FVA cells are capable of enucleating in suspension culture in vitro, indicating that associations with an extracellular matrix or accessory cells are not required for enucleation to occur. The morphology of FVA cells undergoing enucleation is nearly identical to erythroblasts enucleating in vivo. The nucleus is segregated to one side of the cell and then appears to be pinched off resulting in an extruded nucleus and reticulocyte. The extruded nucleus is surrounded by an intact plasma membrane and has little cytoplasm associated with it. Newly formed reticulocytes have an irregular shape, are vacuolated and contain all cytoplasmic organelles. The spatial distribution of several cytoskeletal proteins was examined during the maturation process. Spectrin was found associated with the plasma membrane of FVA cells at all stages of maturation but was segregated entirely to the incipient reticulocyte during enucleation. Microtubules formed cages around nuclei in immature FVA cells and were found primarily in the incipient reticulocyte in cells undergoing enucleation. Reticulocytes occasionally contained microtubules, but a generalized diffuse distribution of tubulin was more common. Vimentin could not be detected at any time in FVA cell maturation. Filamentous actin (F-actin) had a patchy distribution at the cell surface in the most immature erythroblasts, but F-actin bundles could be detected as the cells matured. F-actin was found concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts. Newly formed reticulocytes exhibited punctate actin fluorescence whereas extruded nuclei lacked F-actin. Addition of colchicine, vinblastine, or taxol to cultures of FVA cells did not affect enucleation. In contrast, cytochalasin D caused a complete inhibition of enucleation that could be reversed by washing out the cytochalasin D. These results demonstrate that F-actin plays a role in enucleation while the complete absence of microtubules or excessive numbers of polymerized microtubules do not affect enucleation.