AbstractLong‐chain N‐acylethanolamines (NAE), including the endocannabinoid, anandamide, accumulate in mammalian tissues under a variety of pathological conditions. They have also been shown to inhibit the growth of various cancer cell lines in vitro. Here, we report the presence, in widely differing amounts (3.88–254.46 pmol/μmol lipid P), of NAE and their precursor phospholipids in various human tumors and some adjacent unaffected tissue. Anandamide ranged from 1.5 to 48% of total NAE, and incubation of tissue homogenates suggested possible NAE biosynthesis by both the established transacylation‐phosphodiesterase pathway via N‐acyl PE and by direct N‐acylation of ethanolamine.