1 Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs for the treatment of many disease conditions. In the present study, we investigated the effects of the synthetic CB(2) ligand, JWH-133 on the production of interleukins (ILs), IL-12 and IL-10 by lipopolyssacharide (LPS) or Theiler's virus (TMEV)-activated macrophages. 2 JWH-133 evoked a concentration-related inhibition (10 nM-5 microM) of LPS/IFN-gamma induced IL-12p40 release. The effect of JWH-133 (100 nM) was significantly blocked by the CB2 antagonist SR-144528 (1 microM). Macrophages infected with TMEV increased IL-12p40 production and activation of CB2 receptors by JWH-133 (100 nM) inhibited it. 3 The inhibitory effect of JWH-133 (100 nM) on IL-12p40 production may involve extracellular-regulated kinase (ERK1/2) signaling: (i) JWH-133 induced a greater and sustained activation of ERK1/2 kinase in comparison with the level of activation observed following LPS; (ii) the inhibition of ERK1/2 by the specific inhibitor PD98059 increased LPS-induced IL-12p40 production in the presence or absence of JWH-133 suggesting a negative regulation of ERK pathway on IL-12p40 biosynthesis. 4 Activation of CB2 receptors by JWH-133 (10 nM-5 microM) enhanced IL-10 release by LPS/IFN-gamma-activated macrophages and addition of SR144558 (1 microM) totally blocked the effect of JWH (100 nM). 5 Inhibition of ERK by PD98059 significantly suppressed IL-10 production by LPS-activated macrophages. Endogenous IL-10 plays a modulatory role in IL-12 production. Blocking IL-10 with neutralizing antibody resulted in increased IL-12p40 secretion by LPS-activated macrophages in the absence or presence of JWH-133. In contrast, the addition of exogenous mIL-10 reduced the secretion of IL-12p40 in response to LPS.