Arachidonic acid ethanolamide (anandamide) was previously identified as an endogenous cannabinoid receptor ligand. Given that many arachidonic acid-derived mediators exist as families, we hypothesized additional unsaturated fatty acid ethanolamides might bind the cannabinoid receptor. We synthesized linoleylethanolamide, linolenylethanolamide, homo-γ-linolenylethanolamide (5), and 7,10,13,16-docosatetraenylethanolamide (6), then used TLC and GC-MS to compare with porcine brain extracts. Compounds 5 and 6 were identified in brain. Both are oily, with NMR spectra similar to anandamide but differing in vinyl/doubly allylic protons. GC-MS showed they form parent/dehydrated (2-oxazoline) peak pairs, with retention times distinct from anandamide. EI/CI spectra of brain-derived and synthetic 5/6 were identical. In ligand binding assays, 5 and 6 competitively displaced [³H]HU-243 from rat synaptosomal membranes, with Ki values (53.4±5.5 nM and 34.4±3.2 nM) similar to anandamide (52±1.8 nM) and Δ⁹-THC (46±3 nM). This identifies two new brain-derived cannabinoid receptor-binding ethanolamides, showing anandamide is not the sole member of this mediator class; further study is needed on their biological activity differences.