Influenza A virus is highly pleomorphic with particles exhibiting either spherical or filamentous morphology. The mechanisms behind this pleomorphism and its importance in viral pathogenesis are not clearly understood. We have observed that budding of filamentous influenza A/Udorn virus particles can be readily visualized by immunofluorescence microscopy. Filamentous particle formation was inhibited by treatment of cells with the anti-M2 14C2 antibody, but was not inhibited with the isotype identical 5C4 anti-M2 antibody or by anti-neuraminidase antibody. To further explore the viral determinants of filamentous particle formation, we investigated the morphology and growth characteristics of three variants of A/Udorn/72 virus, which had previously been selected for their resistance to growth inhibition by the 14C2 anti-M2 monoclonal antibody. Two of the variant viruses, 5A and 10A, contain single amino acid substitutions in the cytoplasmic domain of the M2 protein, whereas the 1A variant contains a single amino acid substitution in the viral matrix protein, M1. Variants 5A and 10A both were found to retain the filamentous particle phenotype found in the parental strain A/Udorn/72, and the production of filamentous virions by both variants was resistant to inhibition by the 14C2 antibody. However, immunofluorescence and electron microscopy revealed that the variant 1A was composed almost exclusively of spherical particles. The 1A variant displayed higher viral yields and a larger plaque size than the filamentous viruses. In addition, we separated distinct populations highly enriched in spherical or filamentous particles by velocity gradient centrifugation. Analysis of the protein compositions of these particles revealed that the NP:M1 or NP:HA ratios in filamentous particles were significantly lower than in spherical particles, but the filaments have higher levels of NP per particle. The spherical and filamentous particles were found to have similar specific infectivity. These results indicate that the filamentous morphology of the A/Udorn virus depends upon the matrix (M1) and/or M2 proteins.