Caffeine, a nonspecific adenosine receptor (AR) antagonist is widely used to treat apnea of prematurity. Because adenosine modulates multiple biologic processes including inflammation, we hypothesized that AR blockade by caffeine would increase cytokine release from neonatal monocytes. Using cord blood monocytes (CBM), we investigated 1) the changes in AR mRNA profile by real time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and protein expression (western blot) after in vitro culture, caffeine or lipopolysaccharide (LPS) exposure, and 2) the modulation of cytokine release and cyclic adenosine monophosphate (cAMP) production by enzyme-linked immunosorbent assay (ELISA) induced by caffeine and specific AR antagonists: DPCPX(A1R), ZM241385(A2aR), MRS1754(A2bR), and MRS1220(A3R). After 48 h in culture, A2aR and A2bR gene expression increased 1.9 (p = 0.04) and 2.5-fold (p = 0.003), respectively. A1R protein expression directly correlated with increasing LPS concentrations (p = 0.01), with minimal expression preexposure. Only caffeine (50 microM) and DPCPX (10 nM) decreased tumor necrosis factor-alpha (TNF-alpha) release from LPS activated-CBM by 20 and 25% (p = 0.01) and TNF-alpha gene expression by 30 and 50%, respectively, in conjunction with a > or =2-fold increase in cAMP (p < 0.05). AR blockade did not modulate other measured cytokines. The induction of A1R after LPS exposure suggests an important role of this receptor in the control of inflammation in neonates. Our findings also suggest that caffeine, via A1R blockade, increases cAMP production and inhibits pretranscriptional TNF-alpha production by CBM.