The effects of activating protein kinase C (PKC) with indolactam V (Indo-V) and 1,2-dioctanoyl-sn-glycerol (DOG) on smooth muscle intracellular Ca²⁺concentrations ([Ca²⁺]_i) and arterial diameter were determined using ratiometric Ca²⁺ imaging and video edge detection of pressurized rat posterior cerebral arteries. Elevation of intraluminal pressure from 10 to 60 mmHg resulted in an increase in [Ca²⁺]_ifrom 74 ± 5 to 219 ± 8 nM and myogenic constriction. Application of Indo-V (0.01–3 μM) or DOG (0.1–30 μM) induced constriction and decreased [Ca²⁺]_ito 140 ± 11 and 127 ± 12 nM, respectively, at the highest concentrations used. In the presence of Indo-V, the dihydropyridine Ca²⁺-channel-blocker nisoldipine produced nearly maximum dilation and decreased [Ca²⁺]_ito 97 ± 7 nM. In α-toxin-permeabilized arteries, the constrictor effects of Indo-V and DOG were not observed in the absence of Ca²⁺. Both PKC activators significantly increased the degree of constriction of permeabilized arteries at different [Ca²⁺]_i. We conclude that 1) Indo-V- or DOG-induced constriction of pressurized arteries requires Ca²⁺ influx through voltage-dependent Ca²⁺ channels, and 2) PKC-induced constriction of pressurized rat cerebral arteries is associated with a decrease in [Ca²⁺]_i, suggesting an increase in the Ca²⁺sensitivity of the contractile process.