Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-beta(1), and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A(2B)R. Based on this fact, we further purified CCFCs from A(2B)R-deficient mice and demonstrated that A(2B)R is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-beta functions downstream of the A(2B)R to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A(2B)R signaling and offer a potential target for prevention and treatment of penile fibrosis, a dangerous complication seen in priapism.-Wen, J., Jiang, X., Dai, Y., Zhang, Y., Tang, Y., Sun, H., Mi, T., Phatarpekar, P. V., Kellems, R. E., Blackburn, M. R., Xia, Y. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A(2B) adenosine receptor signaling.