The effects of the endogenous, platelet-derived vasoactive compounds, diadenosine tetraphosphate (AP 4A), diadenosine pentaphosphate (AP 5A), and diadenosine hexaphosphate (AP 6A) on the vasoconstriction of isolated rat renal resistance vessels and rat aortic strips were measured using a vessel myograph. In addition, the effects of AP 4A, AP 5A, and AP 6A on the cytosolic free calcium concentration ([Ca 2+] i) were evaluated in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent dye technique. Diadenosine polyphosphates dose-dependently increased the force of renal resistance vessels and isolated aortic strips. The administration of 10 μmol/L AP 4A, AP 5A, or AP 6A significantly increased the force of isolated renal resistance vessels by 3.48 ± 0.43 mN (n = 8), 2.14 ± 0.40 mN (n = 12), or 2.70 ± 0.31 mN (n = 11, each P < .01 compared with resting tension), respectively. The administration of 10 μmol/L AP 4A, AP 5A, or AP 6A significantly increased the force of isolated aortic strips by 2.45 ± 0.97 mNewton (n = 10), 2.70 ± 0.30 mN (n = 6), or 1.48 ± 0.20 mN (each P < .01 compared with resting tension), respectively. The administration of 10 μmol/L AP 4A, AP 5A, or AP 6A significantly increased [Ca 2+] i in VSMC to a peak concentration of 314 ± 60 nmol/L (n = 6), 247 ± 25 nmol/L (n = 15), or 332 ± 100 nmol/L (n = 5), respectively (each P < .01 compared with resting value). Both the diadenosine polyphosphate-induced vasoconstriction and [Ca 2+] i increase was significantly reduced in the absence of extracellular calcium or after administration of a specific inhibitor of P2 purinoceptors. It is concluded that diadenosine polyphosphates increase [Ca 2+] i and hence cause vessel constriction.