The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence (139)RSSEDKSTQTTGR(151). Site-directed mutagenesis of residues D(143) and Q(147) to an A residue abolished binding to LC8. The P-LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140-150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.