The effect of ATP in human and rabbit corpus cavernosum (CC) smooth muscle was investigated. Strips of human CC were vertically mounted in an organ bath and the tonic tension was recorded. ATP (0.1-3 mM) induced a concentration-dependent relaxant effect, with a pD₂ value of 3.01 +/− 0.3. The purine-induced relaxation was not affected by L-NAME (100 micro M). In rabbit CC, ATP also induced a concentration-dependent relaxation, which was not influenced by L-NAME or by indomethacin (3 micro M), with a pD₂ value of 3.1 +/− 0.4. The ATP-induced relaxant effect in rabbit CC was increased by both the inhibitor of adenosine reuptake, dipyridamole (3 micro M) and by the inhibitor of adenosine deaminase, EHNA (0.3 micro M). Moreover CGS 15943 (3 micro M), an A_2a adenosine antagonist, reduced the ATP-induced relaxation. UTP was not able to produce relaxation. The two ATP analogues 2-methylthioATP and alpha, beta-methylene ATP were able to induce relaxation in rabbit CC, with the following order of potency: 2-methylthioATP > ATP > alpha, beta-methylene ATP thus suggesting a role for P_2y receptors. However, reactive blue (500 micro M), an unspecific P_2y antagonist, did not modify the ATP relaxant response. The inhibition of phospholipase C by U73122 (3 micro M) and of the endoplasmic reticulum Ca²⁺ ATPase by thapsigargin (1 micro M) did not modify the ATP-induced relaxation. The P_2x specific antagonist PPADS (30 micro M) and suramine (500 micro M) were not able to modify the ATP relaxation either in the absence or presence of CGS 15943 (3 micro M). These results confirm that ATP acts as a potent and NO-independent relaxant agent of human and rabbit CC. Our findings also show that the ATP effect is partially attributable to the metabolic breakdown of ATP to adenosine, which acts through A_2a receptor stimulation, but is also due to a direct stimulation of P₂ receptors that are different from the classical P_2y and P_2x receptor subtypes for ATP.