Free-floating aquatic plants are useful in studies of plant biochemistry. Their value lies in the fact that they are naturally adapted to growth on liquid media which can be precisely manipulated. Critical biochemical studies require axenic plants free of contaminating organisms which contribute unwanted chemical products or bind/metabolize added compounds. Axenic plants of Lemna, Spirodela, and Wolffiella species have been available for some time. Access to axenic cultures of the free-floating aquatic plant, Pistia stratiotes L., Araceae, (water lettuce) would provide a model more representative of higher and terrestrial plants (leaves, stems, stolons, multiple roots). Our choice of P. stratiotes focuses on its value as a model to study oxalic acid formation and calcium regulation as related to calcium oxalate production. To this end, we have developed axenic Pistia cultures and compared non-axenic plants grown in greenhouse conditions to the axenic plants in terms of organic acid content and morphology/anatomy. Axenic Pistia were comparable to greenhouse-grown plants, the only difference being the smaller size of axenic plants. This study demonstrates the suitability of axenic Pistia for biochemical research and also indicates that such plants will be very useful in studies of bioremediation and other processes in aquatic systems.