We performed studies to determine the effect of extracellular ATP on the intracellular Ca2+ concentration ([Ca2+](i)) in freshly isolated microvascular smooth muscle cells (MVSMC). Suspensions of preglomerular MVSMC were prepared by enzymatic digestion and loaded with fura 2. Single cells were studied using a microscope-based fluorescence spectrophotometer during superfusion of a physiological salt solution with 1.8 mM Ca2+ and during exposure to similar solutions containing ATP. Under control conditions, baseline [Ca2+](i) averaged 107 ± 6 nM (n = 86 cells from 34 animals). ATP administration elicited concentration-dependent increases in [Ca2+](i). Exposure to ATP concentrations of 1, 10, and 100 μM increased intracellular Ca2+ to peak concentrations of 133 ± 20, 338 ± 37, and 367 ± 35 nM, respectively (P < 0.05 vs. respective baseline). Steady-state [Ca2+](i) increased to 113 ± 15, 150 ± 16 (P < 0.05 vs. baseline), and 180 ± 12 nM (P < 0.05 vs. baseline) for the same groups. The [Ca2+](i) response to ATP was also assessed in the absence of extracellular Ca2+ and during blockade of L-type Ca2+ channels with diltiazem. In these studies, exposure to 100 μM ATP induced a transient peak increase in [Ca2+](i) with the plateau phase being totally abolished under Ca2+-free conditions and markedly attenuated during Ca2+ channel blockade, respectively. These data indicate that ATP-mediated P2-receptor activation increases [Ca2+](i) in freshly isolated preglomerular MVSMC by stimulating Ca2+ release from intracellular stores, in addition to stimulating the influx of extracellular Ca2+ through voltage-gated L-type Ca2+ channels.