1. Experiments were conducted on cultured renal arterial smooth muscle cells to determine the ability of extracellular ATP to alter cytosolic calcium concentration and to determine the mechanisms by which this effect occurs. 2. ATP (100 μmol/L) caused the fluorescence ratio of fura-2 to increase from a control value of 1.06 ± 0.05 to 2.06 ± 0.13 (P < 0.01) before stabilizing at a sustained level of 1.35 ± 0.04 (n = 8;P <0.05). 3. Removal of extracellular calcium from the bathing medium resulted in an attenuation of the initial response to 100 μmol/L ATP with cell fluorescence increasing from 1.16 ± 0.18 to 1.44 ± 0.18 ratio units (n = 5). Furthermore, the initial increase in fluorescence ratio rapidly declined to 1.02 ± 0.06, indicating that an influx of extracellular calcium is required to sustain the increase in fura-2 fluorescence. 4. Depletion of intracellular calcium pools with thapsigargin prevented the increase in fura-2 fluorescence evoked by ATP. 5. These data suggest that ATP-mediated increases in cytosolic calcium in cultured renal arterial smooth muscle cells involve calcium release from the thapsigargin-sensitive, intracellular pool in conjunction with calcium influx from the extracellular medium.