Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor ␣ mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB 2 ) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB 2 receptor more strongly than the endogenous cannabinoids. The K i values of A1 and A2 (CB 2 ϳ60 nM; CB 1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [ 3 H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB 2 , directed by H-bonding and -interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB 2 and CB 1 . A1 and A2 elevated total intracellular Ca 2؉ in CB 2 -positive but not in CB 2 -negative promyelocytic HL60 cells, an effect that was inhibited by the CB 2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB 2 K i >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB 2 -dependent manner. A1, A2, anandamide, the CB 2 antagonist SR144528 (K i <10 nM), and also the non-CB 2 -binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor ␣, IL-1, and IL-12p70 expression (5-500 nM) in a CB 2 -independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB 2 binding.