Anandamide loses its cannabimimetic activities upon hydrolysis to arachidonic acid and ethanolamine. So far the anandamide hydrolyzing activity widely distributed in mammalian organs has been attributed exclusively to an enzyme referred to as anandamide amidohydrolase with an optimum pH around 9. We found another enzyme hydrolyzing anandamide in a human megakaryoblastic cell line (CMK). The enzyme present in the 12,000 x g pellet of the cell homogenate was solubilized by freeze-thaw. The solubilized enzyme showed an optimal pH around 5, and was almost inactive at alkaline pH. The enzyme activity was increased by the addition of dithiothreitol. In contrast, anandamide amidohydrolase of RBL-1 cells was mostly insoluble even after freeze-thaw, showed an optimal pH at 9, and was not affected by dithiothreitol. Furthermore, the enzyme of CMK cells was much less sensitive to phenylmethylsulfonyl fluoride and methyl arachidonoyl fluorophosphonate potently inhibiting anandamide amidohydrolase, and effectively hydrolyzed palmitoylethanolamide, which was a poor substrate for anandamide amidohydrolase. Thus, the enzyme of CMK cells is distinguishable from anandamide amidohydrolase.