Plants are emerging as a promising alternative to conventional platforms for the large-scale production of recombinant proteins. This field of research, known as molecular farming, is developing rapidly and several plant-derived recombinant proteins are already in advanced clinical trials. However, the full potential of molecular farming can only be realized if we gain a fundamental understanding of biological processes regulating the production and accumulation of functional recombinant proteins in plants. Recent studies indicate that species- and tissue-specific factors as well as plant physiology can have a significant impact on the amount and quality of the recombinant product. More detailed comparative studies are needed for each product, including the analysis of expression levels, biochemical properties, in vitro activity and subcellular localization. In this review we include the first results from an extensive comparative study in which the highly glycosylated enzyme phytase (from the fungus Aspergillus niger) was produced in different plant species (including tobacco and the model legume Medicago truncatula). Special emphasis is placed on M. truncatula, whose leaves accumulated the highest levels of active phytase. We discuss the potential of this species as a novel production host.