Two vesicular stomatitis virion proteins, NS and M, are phosphorylated in vivo before packaging and in vitro during the transcription process carried out with disrupted virions. Phosphorylation of NS is thought to be essential for transcription but which of the many acceptor sites is or are involved in this function and which protein kinase(s) is responsible still need to be resolved. We recently reported that the virion-associated kinase which modifies M protein is most likely a different enzyme than that phosphorylating NS (Beckes et al., Virology 169, 161-171 1989). Here we present additional evidence for the presence of distinct enzymes modifying M vs NS substrates and also show that at least two distinct kinase activities modify NS protein. Each of the three activities displayed different optimum reaction conditions, phosphate donor preferences, and sensitivity to inhibition by N-ethylmaleimide.