Certain temperature-sensitive (ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65gag, at 39 degrees, the nonpermissive temperature. At 39 degrees, virions released from cells infected with the various ts mutants also contained elevated levels of Pr65gag relative to virions released at 33 degrees, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr65gag accumulated in the cytoskeleton-enriched fraction at 39 degrees but not at 33 degrees. During steady-state labeling, as much as half of the intracellular Pr65gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39 degrees. At permissive temperature only 10-15% of the intracellular Pr65gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65gag in the cytoskeleton-enriched cell fraction during a short pulse at 39 degrees, but Pr65gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33 degrees. Based upon these and previous results (Edbauer and Naso, 1983), models for retrovirus assembly are described in which the association of Pr65gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.