We surveyed charge heterogeneity in the polypeptides of three enveloped RNA viruses, Sendai virus, influenza virus (WSN strain), and vesicular stomatitis virus (VSV). Isoelectric focusing in polyacrylamide gel was followed by electrophoresis in a second dimension after denaturation of the polypeptides with sodium dodecyl sulfate. Nucleocapsid polypeptides P and NP of Sendai virus exhibited charge heterogeneity which may correspond to various extents of post-translational phosphorylation ( R. A. Lamb and P. W. Choppin, 1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology 81, 382–397. Nucleocapsid polypeptide N of VSV was more homogeneous, whereas the NP polypeptide of influenza virus appeared to be too basic (isoelectric point greater than 8.0) to be resolved in the isoelectric focusing system employed. Glycosylated envelope polypeptides of all three viruses separated into multiple (3 to 8) acidic species with isoelectric points in the range of 4 to 5 for VSV glycopolypeptide G and 5 to 6 for the glycopolypeptides of Sendai virus and influenza virus. Although some of the heterogeneity in VSV glycopolypeptide G may stem from variations in content of N-acetyl neuraminic acid (NANA), a different basis for the heterogeneity is suggested by failure of extensive neuraminidase treatment to abolish it. Moreover, NANA is removed from the glycopolypeptides of Sendai virus and influenza virus by virus-specified neuraminidases. A possible relationship between content of amino sugars (other than NANA) and charge heterogeneity was suggested by the finding that the amount of radioactive glucosamine incorporated biosynthetically into G of VSV and HN of Sendai virus was greater in the more electropositive species. The functional consequences of this variety of post-translational modifications remain to be determined.