The adenosine (ADO) receptor in NIH 3T3 fibroblasts was characterized using a series of adenosine agonists and selected xanthine and non-xanthine antagonists. The ADO receptor elicited accumulations of cyclic AMP in intact NIH 3T3 fibroblasts and caused activation of adenylate cyclase in membrane preparations. The receptor had characteristics of the A2b subtype of adenosine receptor. ADO analogs had relatively high EC50 values at the receptor and were antagonized competitively by xanthines. The rank order of potency for adenosine analogs in NIH 3T3 fibroblasts for cyclic AMP accumulation was: NECA > 2-ClADO > R-PIA >> CV1808, CGS 21680. The EC50 for 2-ClADO was 4.3 microM in intact cells and 15 microM in membrane preparations. All ADO analogs were more potent at the A2a receptor of pheochromocytoma PC12 membranes than at the A2b receptor of fibroblast NIH 3T3 membranes. Structure-activity relationships suggested that the regions of interaction with 5'- and N6-substituents of ADO were similar for both the PC12 A2a and NIH 3T3 A2b receptor. However, ADO analogs with large substituents in the 2'-position, such as 2-cyclohexylethoxy ADO and CGS 21680, were highly selective for the A2a receptor. All ADO analogs tested were stimulatory to adenylate cyclase at the NIH 3T3 A2b receptor, including 5'-methylthioADO, which was a weak partial agonist. A series of xanthine antagonists were not selective for the NIH 3T3 A2b versus the PC12 A2a receptor. In all cases, xanthines were more potent as antagonists in the intact NIH 3T3 cells than in NIH 3T3 membranes. In a series of non-xanthine antagonists, most compounds were equipotent or slightly more potent at the A2a receptor except for alloxazine, which was approximately 9-fold selective for the A2b receptor.