The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype. However, several lines of evidence indicated that this growth-promoting effect was cannabinoid receptor-independent. First, the potent synthetic cannabinoid agonist CP 55940, which displays high affinity for both receptors, was inactive in this model. Second, SR 141716A and SR 144528, which are potent and specific antagonists of CB1 and CB2 receptors respectively, were unable, alone or in combination, to block the anandamide-induced effect. Third, inactivation of both receptors by pretreatment of cells with pertussis toxin did not affect the potentiation of cell growth by anandamide. These data demonstrated that neither CB1 nor CB2 receptors were involved in the anandamide-induced effect. Moreover, using CB2-transfected Chinese hamster ovary cells, we demonstrated that after complete blockade of the receptors by the specific antagonist SR 144528, anandamide was still able to strongly stimulate a mitogen-activated protein (MAP) kinase activity, clearly indicating that the endogenous cannabinoid can transduce a mitogenic signal in the absence of available receptors. Finally, arachidonic acid, a structurally related compound and an important lipid messenger without known affinity for cannabinoid receptors, was shown to trigger MAP kinase activity and cell growth enhancement similar to those observed with anandamide. These findings provide clear evidence for a functional role of anandamide in activating a signal transduction pathway leading to cell activation and proliferation via a non-cannabinoid receptor-mediated process.