The mechanisms responsible for the uptake and cellular processing of the endogenous cannabinoid anandamide are not well understood. We propose that anandamide uptake may occur via a caveola/lipid raft-related endocytic process in RBL-2H3 cells. Inhibitors of caveola-related (clathrin-independent) endocytosis reduced anandamide transport by approximately 50% compared with the control. Fluorescein derived from fluorescently labeled anandamide colocalized with protein markers of caveolae at early time points following transport. In this study, we have also identified a yet unrecognized process involved in trafficking events affecting anandamide following its uptake. Following uptake of [(3)H]anandamide by RBL-2H3 cells, we found an accumulation of tritium in the caveolin-rich membranes. Inhibitors of both anandamide uptake and metabolism blocked the observed enrichment of tritium in the caveolin-rich membranes. Mass spectrometry of subcellular membrane fractions revealed that the tritium accumulation observed in the caveolin-rich membrane fraction was not representative of intact anandamide, suggesting that following metabolism by the enzyme fatty acid amide hydrolase (FAAH), anandamide metabolites are rapidly enriched in caveolae. Furthermore, HeLa cells, which do not express high levels of FAAH, showed an accumulation of tritium in the caveolin-rich membrane fraction only when transfected with FAAH cDNA. Western blot and immunocytochemistry analyses of RBL-2H3 cells revealed that FAAH was localized in intracellular compartments distinct from caveolin-1 localization. Together, these data suggest that following uptake via caveola/lipid raft-related endocytosis, anandamide is rapidly metabolized by FAAH, with the metabolites efficiently recycled to caveolin-rich membrane domains.