Wheat low-molecular-weight-glutenin and alpha-gliadin were accumulated in the endoplasmic reticulum and formed protein body-like structures in tobacco cells, with the participation of BiP chaperone. Possible interactions between these prolamins were investigated. Wheat prolamins are the major proteins that accumulate in endosperm cells and are largely responsible for the unique biochemical properties of wheat products. They are accumulated in the endoplasmic reticulum (ER) where they form protein bodies (PBs) and are then transported to the storage vacuole where they form a protein matrix in the ripe seeds. Whereas previous studies have been carried out to determine the atypical trafficking pathway of prolamins, the mechanisms leading to ER retention and PB formation are still not clear. In this study, we examined the trafficking of a low-molecular-weight glutenin subunit (LMW-glutenin) and alpha-gliadin fused to fluorescent proteins expressed in tobacco cells. Through transient transformation in epidermal tobacco leaves, we demonstrated that both LMW-glutenin and alpha-gliadin were retained in the ER and formed mobile protein body-like structures (PBLS) that generally do not co-localise with Golgi bodies. An increased expression level of BiP in tobacco cells transformed with alpha-gliadin or LMW-glutenin was observed, suggesting the participation of this chaperone protein in the accumulation of wheat prolamins in tobacco cells. When stably expressed in BY-2 cells, LMW-glutenin fusion was retained longer in the ER before being exported to and degraded in the vacuole, compared with alpha-gliadin fusion, suggesting the involvement of intermolecular disulphide bonds in ER retention, but not in PBLS formation. Co-localisation experiments showed that gliadins and LMW-glutenin were found in the same PBLS with no particular distribution, which could be due to their ability to interact with each other as indicated by yeast two-hybrid assays.