The ability of adenosine (ADO) to inhibit proliferation and protein synthesis (in particular, collagen synthesis) in cardiac fibroblasts (CF) may ameliorate adverse cardiac remodeling and fibrosis seen in heart failure patients. However, little is known about the signaling pathways that ADO may modulate in CF to alter cell phenotype. Accordingly, this study was designed to identify ADO receptors (AR) and the signaling pathways linked to them in primary cultures of adult rat CF. Quantitative RT-PCR data indicate that the mRNAs for all four known ARs (A₁R, A_2aR, A_2bR, and A₃R) are present in rat CF, with a greater prevalence of A₂ receptor subtypes. No coupling of AR to the G_q-phospholipase C signaling pathway or to mobilization of calcium is measurable. Studies using subtype specific agents imply that the A_2aR and A_2bR couple to G_s-adenylyl cyclase and A₁R couple weakly to G_i-adenylyl cyclase. 2-Chloroadenosine, 5'-N-ethylcarboxamidoadensoine, and other agents that elevate cellular cAMP stimulate extracellular signal-regulated kinase 1/2 activity in a pertussis toxin-insensitive manner. We conclude that a combination of cAMP-dependent signals generated via A_2a and A_2b receptors likely mediate ADO signaling in adult rat CF.