Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A₂ (A_2a and A_2b) receptor (A_2aR and A_2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A_2a and/or A_2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A_2aR or A_2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A₁R, A_2bR, A_2aR, and A₃R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A₂R and A_2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A_2aR or A_2bR. Overexpression of A_2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A_2bR yielded increases in protein and collagen synthesis. In contrast, A_2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A_2bR are critically involved in ADO-mediated inhibition of CF functions.