1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [3H]-adenine pre-labelling methodology. 2. Adenosine analogues showed the following rank order of potency (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) > or = adenosine (4.19)= N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A2A-selective agonist CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 microM. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76+/-8% NECA response), 2-chloroadenosine (70+/-6%) and APNEA (40+/-3%) were significantly reduced. 4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93) 8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13). 5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A2B adenosine receptor. 6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A2B subtype.