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The temperature dependency of anandamide uptake into cells implies an active mechanism but this is still a matter of considerable debate. We have therefore re-examined the temperature-sensitive uptake of anandamide in ND7/23 mouse neuroblastoma × rat dorsal root ganglion neurone hybrid cells and RBL2H3 rat basophilic leukaemia cells.Cellular uptake of [3H] anandamide was measured in the presence of bovine serum albumin at different incubation temperatures and times. Rates of uptake were also measured in wells alone. Free anandamide concentrations were calculated by published methods.Anandamide showed a time-dependent saturable uptake into ND7/23 cells. The uptake was greater at 37°C than at 4°C for a given added anandamide concentration following a 5 min incubation. However, this temperature-dependency reflected temperature-dependent effects on the concentration of anandamide available for uptake, rather than the uptake process itself. A similar conclusion could be drawn for the rapid (˜1 min) uptake of anandamide into RBL2H3 cells. In contrast, re-analysis of published data for P19 cells indicated a clear temperature-dependency of the uptake at long (15 min) incubation times. The level of anandamide retained by wells alone provided a better measure of free anandamide concentrations than calculated values.ND7/23 cells may be a useful model system for the study of anandamide uptake. The temperature-dependent uptake of anandamide may reflect effects on free anandamide concentrations rather than on the uptake process itself.