The role of the sarcoplasmic reticulum (SR) in regulating myogenic tone and [Ca2+]i was examined with ryanodine and cyclopiazonic acid (CPA) in the rat skeletal muscle arteriole (A(sk)) and mesenteric arteriole (Ams). Arterioles were cannulated at both ends to control luminal pressure in a tissue bath. Luminal diameter was measured with a video-monitored microscopic system. Fura 2-AM was loaded to measure [Ca2+]i using the fluorescence intensity ratio at excitation wavelengths of 340 to 380 nm (F340/380). The myogenic response (luminal pressure was increased from 40 to 100 mm Hg) and the intrinsic tone at 40 mm Hg were observed in A(sk) but not in Ams. Ryanodine (10(-5) M decreased the steady-state diameter of A(sk) from 138 +/- 8 to 85 +/- 9 microns (P < .05) and increased the F340/380 ratio; these effects were reversed by nifedipine or Ca(2+)-free solution. Ryanodine shifted the [Ca2+]o-contraction response curve upward. CPA (10(-5) M) also decreased the steady-state diameter of A(sk) from 131 +/- 7 to 98 +/- 11 microns (P < .05). In contrast, Ams responded to neither ryanodine nor CPA. Caffeine-induced contractions were significantly reduced by either ryanodine or CPA in both arterioles. These results indicate that SR dysfunction increased the susceptibility of the arteriolar tone to [Ca2+]o and enhanced the tone of A(sk). In conclusion, the SR function may play a critical role in regulating [Ca2+]i and the intrinsic tone of A(sk) that was myogenically active at physiological luminal pressure.