Abstract The pharmacokinetic (PK) parameters of artesunate, artemether and their metabolites dihydroartemisinin (DHA) and dihydroartemisinin-glucuronide (DHA-glucuronide) were determined in sheep naturally infected with Fasciola hepatica. Sheep were treated either with artesunate (intramuscular (i.m.): 40 and 60mg/kg) or artemether (i.m.: 40 and 160mg/kg; oral: 80mg/kg). Blood samples were withdrawn at selected time points post treatment and the artemisinins were quantified in plasma by liquid chromatography and tandem mass spectrometry (LC–MS/MS). The in vitro effect of the metabolites against F. hepatica was investigated using a phenotype-based assay and scanning electron microscopy (SEM). Following artesunate applications (40 and 60mg/kg), comparable Cmax (maximal plasma concentration) and AUCs (area under the plasma concentration–time curve) were observed for artesunate (Cmax: 8.4×103 and 9.4×103ng/ml; AUC: 6.9×105 and 9.7×105ngmin/ml), DHA (Cmax: both 2.4×103ng/ml; AUC: 3.7×105 and 5.0×105ngmin/ml), and DHA-glucuronide (Cmax: 1.7×104 and 1.6×104ng/ml; AUC: 2.6×106 and 3.3×106ngmin/ml). Mean elimination half-lifes (t1/2) of artesunate, DHA and DHA-glucuronide ranged between 58 and 63min, 94 and 113min, and 89 and 98min, respectively. The i.m. oil-based drug formulation liberated artemether slowly and constant levels of artemether and its metabolites were observed during the entire sampling period (24h). The AUCs of all analytes were significantly higher for the i.m. 160mg/kg dose compared to i.m. 40 and oral 80mg/kg doses (P=0.018). Mean Cmax of artemether (2126 and 426ng/ml) and DHA-glucuronide (3477 and 1587ng/ml) were higher following oral compared to i.m. (160mg/kg) treatments (P>0.068), whereas Cmax of DHA was significantly higher following i.m. applications (P=0.0062). DHA rapidly reduced the viability of F. hepatica in vitro, whereas DHA-glucuronide showed no activity. SEM observations revealed only minor and focal tegumental alterations in few of the DHA treated worms. The calculated PK parameters reflect the anthelmintic activity of artesunate and artemether following different routes of application and will aid in the design of future studies with these drugs.