Autographa californica nuclear polyhedrosis virus (AcMNPV) is a large, double-stranded DNA virus of lepidopteran insects. It replicates in the nucleus of host cells and produces two different infectious forms. One of them buds from the plasma membrane and is responsible for systemic infection in insects and for the spread of infection in cell culture. Earlier we reported that cytochalasin D (CD) inhibited the production of this form of the virus in infected IPLB-SF-21 cells by a mechanism other than the prevention of budding [L. E. Volkman, P. A. Goldsmith, and R. T. Hess (1987). Virology 156, 32-39]. Herein we present evidence that CD interferes with proper nucleocapsid assembly which indicates that microfilaments are involved in this nuclear process. The observation that phalloidin localizes in the nuclei of 24-hr infected cells in the absence, but not in the presence, of CD supports this hypothesis. Viral DNA replication and synthesis of the major capsid antigen and its transport to the nucleus are not inhibited by CD.