Extensive characterization of adenosine receptors expressed by human monocyte-derived dendritic cells (MDDCs) was performed with quantitative polymerase chain reaction, radioligand binding, and calcium signaling. Transcript for the A3 adenosine receptor was elevated more than 100-fold in immature MDDCs compared with monocyte precursors. A3 receptor transcript was substantially diminished, and A2A receptor transcript increased, by lipopolysaccharide maturation of MDDCs. Saturation binding of N(6)-(3-[(125)I]iodo-4-aminobenzyl)-adenosine-5'-N-methyluronamide ([(125)I]AB-MECA) to membranes from immature MDDCs yielded B(max) of 298 fmol/mg of protein and K(D) of 0.7 nM. Competition against [(125)I]AB-MECA binding confirmed the site to be the A3 receptor. Adenosine elicited pertussis toxin-sensitive calcium responses with EC(50) values ranging as low as 2 nM. The order of potency for related agonists was N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) >/= I-AB-MECA > 2Cl-IB-MECA >/= adenosine > 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxyamidoadenosine (CGS21680). The order of efficacy was adenosine >/= CGS21680 > IB-MECA >/= I-AB-MECA > 2Cl-IB-MECA. Calcium responses to 2Cl-IB-MECA and CGS21680, and the lower range of adenosine concentrations, were completely blocked by 10 nM N-(2-methoxyphenyl)-N-[2-(3-pyridyl)quinazolin-4-yl]urea (VUF5574) but not by 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) or 8-cyclopentyl-1,3-dipropylxanthine. Pretreatment with 100 nM 2Cl-IB-MECA eliminated responses to CGS21680 but not to monocyte inhibitory protein-1alpha. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Galphaq-i3 protein, which was required to establish A3-mediated calcium signaling. The pharmacological profile of calcium signaling elicited by adenosine-related agonists in the double-recombinant cells was essentially identical to that obtained from immature MDDCs. Our results provide an extensive analysis of A3-mediated calcium signaling and unequivocally identify immature MDDCs as native expressers of the human A3 receptor.