On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these ''transport inhibitors'' did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier. T he endocannabinoids, including anandamide, are a class of neurotransmitters, similar to ⌬ 9 -tetrahydracannabinol, involved in multiple physiological events including nociception, memory, blood pressure, locomotion, and immunity (for review, see ref. 1). These compounds bind the CB1 and CB2 cannabinoid receptors, which are G i -coupled receptors that modulate ion channels and signal transduction pathways (2-4).Anandamide is readily taken up into cells. The first step of this process has been characterized by several laboratories as a process of facilitated diffusion (for reviews, see refs. 5 and 6). Although an anandamide transporter has never been isolated, its existence is based on an anandamide uptake process that is temperature-dependent, selective, and saturable. In addition, several studies identified compounds that inhibit anandamide accumulation, including N-(4-hydroxyphenyl)arachidonylamide (AM404) and the vanilloids arvanil and olvanil (7-16).After uptake, if fatty acid amide hydrolase (FAAH) is present, anandamide is hydrolyzed to arachidonic acid and ethanolamine (for review, see ref. 17). A recent report investigating the role of FAAH in anandamide metabolism showed that FAAH Ϫ...