Vesicular stomatitis virus contains a phosphorylated NS protein which is necessary along with L protein and RNP template for transcription of mRNA. To further define the structure of the NS protein and its function in transcription and replication, virion NS was purified and separated into two different phosphrylated forms (NSI and NSII) on DEAE-cellulose columns. Cytoplasmic preparations of NS contained one phosphorylated species which eluted from the column in the same place as the virion NSI. When electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing urea, NSI and NSII each resolved into two components, whereas cell NS migrated as a single band. NSI and cell NS exhibited little activity in a reconstituted transcription assay, whereas the more highly phosphorylated NSII was very active in the same system. Addition of NSI or cell NS to a transcription system containing NSII resulted in even higher levels of activity, indicating that the various NS species might have different enzymatic functions.