—The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca²⁺]_i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca²⁺-sensitive dye fura 2. The effect of ATP and UTP on [Ca²⁺]_i was studied on single cells with standard microscope-based fluorescence photometry techniques. Resting [Ca²⁺]_i averaged 80±3 nmol/L (n=219 single cells from 58 dispersions). ATP (100 μmol/L) increased [Ca²⁺]_i to a peak value of 845±55 nmol/L (n=70 single cells from 38 dispersions) before stabilizing at 124±81 nmol/L. Similarly, 100 μmol/L UTP (n=39 single cells from 26 dispersions) stimulated a peak increase in [Ca²⁺]_i of 1426±584 nmol/L before reaching a stable plateau of 123±10 nmol/L. The [Ca²⁺]_i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 μmol/L ATP induced a transient peak increase in [Ca²⁺]_i, with the plateau phase being totally abolished. In contrast, exposure to 100 μmol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca²⁺]_i. The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 μmol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca²⁺]_i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca²⁺]_i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores.