The A_2A adenosine receptor (A_2AAR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A_2AAR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMΦ (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A_2AAR mRNA. LPS-induced increases in mRNA for A_2AAR and TNFα (tumour necrosis factor α) are reduced by 90% in IPMΦ pretreated with the NF-κB (nuclear factor κB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 μM}. In Wehi-3 cells exposed to LPS, A_2AAR and A_2BAR transcripts are elevated by 290- and 10-fold respectively, the A₁AR transcript is unchanged and the A₃AR transcript is decreased by 67%. The induction of A_2AAR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED₅₀ (effective dose) of LPS is 2.3 ng/ml. A_2AAR receptor number, measured by ¹²⁵I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors·cell⁻¹·min⁻¹ to a B_max of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A_2A agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A_2A agonist, ATL146e, to reduce TNFα release from IPMΦ was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A_2AAR, is part of a delayed feedback mechanism initiated through NF-κB to terminate the activation of human and mouse macrophages.