The interaction of the nominal phosphoprotein (NS) and the nucleoprotein (N) of rabies virus (Evelyn Rokitnicki Abelseth strain) was investigated by expressing these proteins in insect cells and in an in vitro coupled transcription-translation system. The N and NS proteins individually expressed in insect cells interacted with each other in vitro and formed complexes at a N:NS ratio similar to those found in rabies virions. In the in vitro transcription-translation system, both the N and NS proteins when synthesized simultaneously formed N-NS complexes which could be immunoprecipitated with either anti-N or anti-NS antibodies. NS mutant proteins with C-terminal deletion of up to 166 amino acids were still able to form complexes with N protein when synthesized simultaneously. However, when the NS mutant proteins and the N protein were synthesized individually and then mixed together, only the intact NS protein and NS mutant protein with 24 amino acids deleted from the C-terminus bound to the N protein, whereas deletion of 47 or more amino acids from the C-terminus of the NS protein resulted in total loss of binding to the N protein. NS mutants with N-terminal deletions of up to 68 amino acids bound to the N protein when synthesized either simultaneously or individually. These results indicate that both the N- and C-terminal domains of the NS protein of rabies virus are involved in the binding to rabies virus N protein but not in a mutually dependent manner. The interaction of the N-terminal domain of the NS protein with N protein occurs only during simultaneous synthesis of both proteins, whereas the C-terminal region of the NS protein can bind to the N protein when these two proteins are synthesized either simultaneously or separately. The two binding sites of the NS protein to N protein might have important functions in regulating virus transcription and replication as well as in virus assembly.