While infectious IMV are formed in the absence of microtubules, we found no evidence for IEV formation, based on electron microscope examination of cells infected in the presence of nocodazole ( Figure 4 ). We did, however, observe IMV particles partially wrapped in trans-Golgi membranes most probably in the process of abortive IEV formation ( Figure 4D ). Given these data, we examined by indirect immuno¯uorescence whether low amounts of IEV particles are formed in the absence of microtubules. However, we could ®nd no evidence for colocalization of the IEV protein markers A36R, A34R or A33R with vaccinia particles formed in the presence of nocodazole ( Figure 5F ). We also found no evidence for IEV formation, based on their ability to nucleate actin tails ( Figure 5O ). As IEV particle assembly involves wrapping by the Golgi apparatus (Schmelz et al., 1994) , we examined the effects of only disrupting this membrane compartment using brefeldin A. We could ®nd no evidence for IEV formation, based on co-localization of IEV protein markers with virus particles and actin tails in cells infected in the presence of brefeldin A ( Figure 5G±I and P±R). Indeed, in brefeldin A-treated cells, the IEV membrane proteins required for assembly were observed in the endoplasmic reticulum and not the trans-Golgi ( Figure 5H ). In summary, our data indicate that the microtubule cytoskeleton is required for ef®cient IMV assembly and is essential for IEV formation.