The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both 2-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of 2-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of 2-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, 2-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions.