In aquatic invertebrates metabolic nitrogenous waste is excreted predominately as ammonia. Very little is known, however, of the underlying mechanisms of ammonia excretion, particularly in freshwater species. Our results indicate that in the non-parasitic freshwater planarian Schmidtea mediterranea ammonia excretion depends on an acidification of the apical unstirred layer of the body surface and consequent ammonia trapping. Buffering of the environment to a pH of 7 or higher decreased excretion rate. Inhibitor experiments suggested further that the excretion mechanism involves the participation of the V-type H+-ATPase and carbonic anhydrase and possibly also the Na+/K+-ATPase and Na+/H+ exchangers (NHEs). Alkalinization (pH 8.5, 2 days) of the environment led to a 1.9-fold increase in body ammonia levels and to a down-regulation of V-ATPase (subunit A) and Rh-protein mRNA. Further, a two day exposure to non-lethal ammonia concentrations (1 mmol L-1) caused a doubling of body ammonia levels and led to an increase in Rh-protein and Na+/K+-ATPase (α-subunit) mRNA expression levels. In-situ hybridization studies indicated a strong mRNA expression of the Rh-protein in the epidermal epithelium. The ammonia excretion mechanism proposed for S. mediterranea reveals striking similarities to the current model suggested to function in gills of freshwater fish.