We have investigated the association of the influenza virus matrix (M1) and nucleoprotein (NP) with the host cell cytoskeletal elements in influenza virus-infected MDCK and MDBK cells. At 6.5 h postinfection, the newly synthesized M1 was Triton X-100 (TX-100) extractable but became resistant to TX-100 extraction during the chase with a t1/2 of 20 min. NP, on the other hand, acquired TX-100 resistance immediately after synthesis. Significant fractions of both M1 and NP remained resistant to differential detergent (Triton X-114, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], octylglucoside) extraction, suggesting that M1 and NP were interacting with the cytoskeletal elements. However, the high-molecular-weight form of the viral transmembrane protein hemagglutinin (HA), which had undergone complex glycosylation, also became resistant to TX-100 extraction but was sensitive to octylglucoside detergent extraction, indicating that HA, unlike M1 or NP, was interacting with TX-100-insoluble lipids and not with cytoskeletal elements. Morphological analysis with cytoskeletal disrupting agents demonstrated that M1 and NP were associated with microfilaments in virus-infected cells. However, M1, expressed alone in MDCK or HeLa cells from cloned cDNA or coexpressed with NP, did not become resistant to TX-100 extraction even after a long chase. NP, on the other hand, became TX-100 insoluble as in the virus-infected cells. M1 also did not acquire TX-100 insolubility in ts 56 (a temperature-sensitive mutant with a defect in NP protein)-infected cells at the nonpermissive temperature. Furthermore, early in the infectious cycle in WSN-infected cells, M1 acquired TX-100 resistance very slowly after a long chase and did not acquire TX-100 resistance at all when chased in the presence of cycloheximide. On the other hand, late in the infectious cycle, M1 acquired TX-100 resistance when chased in either the presence or absence of cycloheximide. Taken together, these results demonstrate that M1 and NP interact with host microfilaments in virus-infected cells and that M1 requires other viral proteins or subviral components (possibly viral ribonucleoprotein) for interaction with host cytoskeletal components. The implication of these results for viral morphogenesis is discussed.