Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65gag with cytoskeletal elements in infected mouse fibroblasts. The Pr65gag associated with Nonidet P-40 (NP-40)-insoluble cytoskeletal structures appears to be subphosphorylated in comparison to NP-40-soluble Pr65gag. The association of Pr65gag with skeletal elements can be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent, or with buffers of high ionic strength. Both the skeleton-associated Pr65gag and its NP-40-soluble counterpart can be labeled with [3H]palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65gag molecules bound to skeletal elements in the infected cell appear to be more stable to proteolytic processing than NP-40-soluble Pr65gag. While the association of Pr65gag with cytoskeleton elements in the cell is neither increased nor decreased by blocking virus assembly and release with interferon, Pr65gag appears to accumulate in the cytoskeleton-enriched fraction of cells chronically infected with a temperature sensitive mutant of R-MuLV (ts 17) when such cells are grown at the nonpermissive temperature. Based on these and other results, we have proposed a model for the active role of cytoskeleton associated Pr65gag in retrovirus assembly.