The association of African swine fever virus (ASFV) with the cytoskeleton was investigated. Immunofluorescent studies of ASFV infected cells with anti-ASFV serum showed a temporal and spatial development of viral inclusions which moved from a peripheral to a perinuclear location and fused to give a single large perinuclear factory. The migration and fusion of viral inclusions was inhibited by colchicine suggesting a function for microtubules in assembly site organization not previously described. Accumulation of virions outside the inclusions and inhibition of viral release was also observed in colchicine treated cells. Viral antigens and structural elements were retained on the cytoskeleton fraction of Triton X-100 extracted cells. Reorganization of cytoskeletal elements around the assembly sites was demonstrated by transmission electronmicroscopy and by immunofluorescent studies using monoclonal antibodies against actin, tubulin and vimentin. Intermediate filaments accumulated around the viral factories, microtubules were greatly decreased in number and microfilaments were reorganized in association with the plasma membrane. Bundles of 15 nm tubules of unknown origin were also observed around the assembly sites. The distribution of viral proteins in soluble, cytoskeleton and detergent insoluble nuclear fractions was studied by pulse-chase experiments with [35S]methionine. SDS-PAGE analysis showed the presence in the cytoskeletal and nuclear fractions of 150, 72, 38, 28, 19 and 15 kDa virus structural proteins which increased after a 5 h chase. Our results indicate a close association of ASFV replication with the cytoskeleton similar to events described during FV3 replication but which differ from those occurring in poxvirus-infected cells.