Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.