The human astrocytoma cell line CCF-STTGI accumulates [3H]2-AG through an Na(+)- and energy-independent process, with a Km of 0.7 +/- 0.1 microM. Non-radioactive 2-AG, anandamide or the anandamide transport inhibitor 4-hydroxyphenyl arachidonamide inhibit [3H]2-AG uptake with half-maximal inhibitory concentrations (IC50) of 5.5 +/- 1.0 microM, 4.2 +/- 0.3 microM and 1.8 = 0.1 microM, respectively. A variety of lipid transport substrates and inhibitors interfere with neither [3H]2-AG nor [3H]anandamide uptake. These results suggest that 2-AG and anandamide are internalized in astrocytoma cells through a common carrier-mediated mechanism. After incubation with [3H]2-AG, radioactivity is recovered in phospholipids, monoacylglycerols (unmetabolized [3H]2-AG), free fatty acids ([3H]arachidonate) and, to a minor extent, diacylglycerols and triacylglycerols. Arachidonic acid (100 microM) and triacsin C (10 microM), an acyl-CoA synthetase inhibitor, prevent incorporation of [3H]arachidonic acid in phospholipids and significantly reduce [3H]2-AG transport. Thus, the driving force for 2-AG internalization may derive from the hydrolysis of 2-AG to arachidonate and the subsequent incorporation of this fatty acid into phospholipids.