Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosinemediated regulation of erectile function is mainly mediated through A 2B AR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A 2A AR knockout, A 2B AR knockout, and A 2A /A 2B AR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A 2A AR and A 2B AR, whereas neither the A 1 AR nor A 3 AR play a role in vascular tone regulation of the PA. In addition, we showed that A 2A AR-and A 2B AR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A 2A ARmediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A 3 AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction.