Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45 degrees from the centre of the tail and are up to 0.74 mu m in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle, Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.