Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.