Cardiac fibroblasts (CFs) play an essential role in remodeling of the cardiac extracellular matrix. Extracellular nucleotide signaling may provoke a profibrotic response in CFs. We tested the hypothesis that physical perturbations release ATP from CFs and that ATP participates in profibrotic signaling. ATP release was abolished by the channel inhibitor carbenoxolone and inhibited by knockdown of either connexin (Cx)43 or Cx45 (47 and 35%, respectively), implying that hypotonic stimulation induces ATP release via Cx43 and Cx45 hemichannels, although pannexin 1 may also play a role. ATP released by hypotonic stimulation rapidly (<10 min) increased phosphorylated ERK by 5-8 fold, an effect largely eliminated by P2Y(2) receptor knockdown or ATP hydrolysis with apyrase. ATP stimulation of P2Y(2) receptors increased α-smooth muscle actin (α-SMA) production, and in an ERK-dependent manner, ATP increased collagen accumulation by 60% and mRNA expression of profibrotic markers: plasminogen activator inhibitor-1 and monocyte chemotactic protein-1 by 4.5- and 4.0-fold, respectively. Apyrase treatment substantially reduced the basal profibrotic phenotype, decreasing collagen and α-SMA content and increasing matrix metalloproteinase expression. Thus, ATP release activates P2Y(2) receptors to mediate profibrotic responses in CFs, implying that nucleotide release under both basal and activated states is likely an important mechanism for fibroblast homeostasis.