Herpes simplex virus type 1 (HSV-1) establishes life-long latent infection in sensory neurones, making it an ideal gene therapy vector to target neuronal cells. To study retrograde transport of HSV-1, capsid and tegument proteins were tested for interactions with the motor cytoplasmic dynein, using a yeast two-hybrid system and in vitro pull down assays. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35) and the homologous 14 kDa dynein light chains RP3 and Tctex1. The functional importance of VP26 for retrograde transport was confirmed by microinjecting recombinant HSV-1 capsids into living cells. Capsids containing VP26 moved closer to the cell nucleus, while capsids without VP26 remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.