Previous research has shown that β-adrenoceptor (β-AR) agonists have potent anti-inflammatory capabilities, e.g. represented by suppression of release of the proinflammatory cytokines. Aim of this research was to determine whether the effects of β-agonists on LPS-induced TNFα and IL-10 release are influenced by their different stereochemistry. In addition, the role of the β-AR subtypes was studied. The effect of two stereoisomers of the selective β2-AR agonist TA2005 [(R,R)- and (S,S)-] on the LPS-induced TNFα and IL-10 release by U937 macrophages was compared. The (R,R)-stereoisomer was 277 times more potent in inhibiting the TNFα release than the (S,S)-form. The (R,R)-stereoisomer also appeared to be more potent in increasing the IL-10 release. In radioligand binding studies the affinity of (R,R)-TA2005 for the β-adrenoceptor was 755 times higher than the (S,S)-TA2005 stereoisomer. In addition, the elevation of intracellular cAMP in U937 cells appeared to be stereoselective: (R,R)-TA2005 was more potent in elevating intracellular cAMP. The effect of both stereoisomers on the LPS-induced TNFα release could almost completely be antagonized by preincubation with the selective β2-AR-antagonist ICI-118551. Further evidence that the effect of the β-agonists is mediated via the β2-adrenoceptor subtype exclusively was acquired by incubation of U937 cells with selective β1- and β3-agonists. None of these receptor subtype agonists showed significant suppressive effect on TNFα release. This study provides additional proof that the anti-inflammatory effects of β2-agonists are mediated via the β2-adrenoceptor and indicates that these effects are highly dependent on the stereoselectivity of the ligand.