Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vdeltaF12L) or A36R (vdeltaA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at approximately 60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vdeltaA36R-infected cells. Moreover, vdeltaA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vdeltaA36R IEV moved in a stop-start manner at speeds consistent with microtubular movement, although the mean length of vdeltaA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.