© 2015 The British Pharmacological Society. Background and Purpose: Palmitoylethanolamide (PEA) is an endogenous congener of anandamide and potentiates its actions at cannabinoid CB1 and CB2 receptors, and at transient receptor potential vanilloid type-1 (TRPV1) channels. The other endocannabinoid, 2-arachidonoylglycerol (2-AG), was recently suggested to act as a TRPV1 channel agonist. We investigated if PEA enhanced levels of 2-A in vitro or in vivo and 2-AG activity at TRPV1 channels. Experimental Approach: Endogenous lipid levels were measured by LC-MS in (i) human keratinocytes incubated with PEA (10-20 μM, 40 min, 6 and 24 h, 37°C); (ii) the blood of spontaneously Ascaris suum hypersensitive beagle dogs given a single oral dose of ultramicronized PEA (30 mg·kg-1, 1, 2, 4 and 8 h from administration); (iii) the blood of healthy volunteers given a single oral dose of micronized PEA (300 mg, 2, 4 and 6 h from administration). Effects of 2-AG at TRPV1 channels were assessed by measuring intracellular Ca2+ in HEK-293 cells over-expressing human TRPV1 channels. Key Results: PEA elevated 2-AG levels in keratinocytes (∼3-fold) and in human and canine plasma (∼2 and ∼20-fold respectively). 2-AG dose-dependently raised intracellular Ca2+ in HEK-293-TRPV1 cells in a TRPV1-dependent manner and desensitized the cells to capsaicin. PEA only slightly enhanced 2-AG activation of TRPV1 channels, but significantly increased 2-AG-induced TRPV1 desensitization to capsaicin (IC50 from 0.75 ± 0.04 to 0.45 ± 0.02 μM, with PEA 2 μM). Conclusions and Implications: These observations may explain why several effects of PEA are attenuated by cannabinoid receptor or TRPV1 channel antagonists. Linked Articles: This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.