Plant cell cultures as platforms for recombinant protein production are favoured over other systems because they combine the benefits of plants (low cost of production, low biosecurity risk, conserved post-translational modifications) with those of controlled cell cultures. However, many factors that affect the correct synthesis and accumulation of the recombinant product still need to be determined; in particular, the trafficking route of the recombinant proteins is poorly understood. Suspension cell cultures of Medicago truncatula Gaertn. have been shown to offer a viable and highly efficient system for the production of a model glycoprotein – phytase from the fungus Aspergillus niger Tiegh. The present study investigated subcellular protein sorting by immunogold detection of recombinant phytase with an electron microscope in four independent Medicago cell cultures expressing phytase. Two lines contained a C-terminal KDEL targeting signal for retention in the endoplasmic reticulum (ER), and the other two did not and were expected to travel through the secretory route; a high and low expressor were examined for each variant of the protein. A differential subcellular location of phytase was found in the four transgenic lines studied. These differences account not only for the version of the recombinant protein (secreted or retained in the ER), but also for the different expression levels.