Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [³H]arachidonic acid from [³H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K_m of 3.7 μM and V_max 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9–10, was abolished by phenylmethylsulfonyl fluoride and was Ca²⁺- and Mg²⁺-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.