Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of intact microtubules (MTs). Treatment of infected cells with sodium azide implicated in an active component of MP transfer to PD. Inhibitors of the actin cytoskeleton (latrunculin and cytochalaxin) significantly inhibited MP targeting while MT inhibitors (colchicine and oryzalin) did not. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. We also show that MP expressed from a TMV vector, did not replace transgenically expressed MP-GFP, confirming that existing MP could not be displaced from the PD pore by newly synthesized MP. When MP-decorated MTs were photobleached, MP-GFP rapidly appeared on the vertices of adjacent cortical ER network and also re-appeared on existing MTs. To examine whether the MP was mobile on MTs, we expressed MP as a fusion to photoactivatable GFP (PAGFP) or to photoswitchable CFP (PSCFP) from a TMV vector. Photoactivated MP-PAGFP remained static on the MTs and MP-PSCFP did not move laterally away from the site of photoconversion over a period in excess of 24 hours. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network whilst excess MP is moved from the cortical ER ot MPs prior to degradation by the host cell.