Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is a large DNA virus that replicates in the cytoplasm of host cells. Actin is a critical component of the host cytoskeleton and also plays an important role in the replication cycle of VACV by promoting virus motility and the spread of infection. As part of its replication cycle, cell-associated enveloped vaccinia virus particles are tethered to the surface of the host cell upon exit and can be released by interactions of the viral protein A36 with a range of host proteins involved in the ARP2/3-complex-dependent actin polymerisation pathway. This results in the formation of actin ‘tails’ which can propel the extracellular enveloped virus particles to neighbouring cells. Recent developments of siRNA and isoform-specific antibodies have enabled an exploration of the functions and relative distribution of the previously poorly understood isoforms of cytoplasmic actin (CYA): beta- and gamma-actin. We have employed these techniques and more to study the role of these two CYA isoforms in vaccinia virus tail morphology and viral release, revealing divergent roles for these proteins. We have also conducted experiments to define the mechanism of this difference in the ARP2/3-complex-associated actin polymerisation machinery.