The herpes simplex virus 1 (HSV-1) tegument protein VP22 has been utilised as a vehicle for trafficking proteins. It has a remarkable property of exiting the cell that is producing it and entering the neighbouring cells, which has been used to deliver therapeutic proteins, p53 and herpes simplex virus thymidine kinase (tk). It has a complex pattern of expression and subcellular localisation. Functions of VP22 include intercellular transport, binding to and bundling of microfilaments, inducing cytoskeleton collapse, nuclear translocation during mitosis, and binding to chromatin and nuclear membrane. The regions of VP22 which contain each of these functions have not been characterised. Finding the region carrying the property of intercellular spread would facilitate enhancement of transport function. By constructing a series of deletion constructs of VP22 tagged by the green fluorescent protein (GFP) we have mapped the functions of VP22 to specific regions in the polypeptide as follows: intercellular transport - aa 81-195; binding and reorganisation of cytoskeleton - aa 159-267; nuclear targeting, inhibition of cytoskeleton collapse - aa 81-121; and nuclear targeting and facilitation of intercellular transport - aa 267-301. Separation of VP22 functions enables focus on the mechanism of VP22-mediated transport and improve the transportation efficiency of VP22.