The purpose of this study was to measure vascular smooth muscle (VSM) cytosolic calcium ([Ca2+]i) during the myogenic response. We examined the temporal and steady-state relationships between lumen diameter and VSM [Ca2+]i in isolated arterioles exposed to step changes in intravascular pressure. We also studied the relationship between step sizes in intravascular pressure and changes in [Ca2+]i. First-order arterioles from the hamster cheek pouch were isolated, cannulated, and pressurized. [Ca2+]i was quantified using the ratio of emitted fluorescence intensity (R340/380) during alternate excitation of fura 2-loaded vessels at 340 and 380 nm. Stepwise increases in transmural pressure elicited corresponding increases in steady-state [Ca2+]i and myogenic constriction. From a common baseline pressure, the initial rise in [Ca2+]i after a step change in pressure was directly related to the magnitude of the step size and of the distension caused by that pressure step. This supports the theory that there is a relationship between the initial distension of the vessel and the initial [Ca2+]i change. Also, increasing the size of the step change in pressure resulted in a greater myogenic response, yet no difference in the steady-state [Ca2+]i was detected, which suggests that Ca2+ is not the principal or only determinant of steady-state constriction. Finally, larger increases in [Ca2+]i do not necessarily augment the myogenic response, which suggests that some minimal rise in [Ca2+]i is required to elicit myogenic vasoconstriction. Collectively, these data suggest the presence of a separate, Ca(2+)-independent regulatory system.