Apart from being released as a co-transmitter from sympathetic nerve terminals, interstitial ATP acting upon P2Y and P2X purinoceptors has been shown to be an important paracrine regulator of renal preglomerular microvascular function [1]. Indeed, inactivation of P2 receptors in renal resistance blood vessels inhibits autoregulatory behaviour [2]. Stimulation of P2X purinoceptors (P2X-Rs) in renal vascular smooth muscle cells (RVSMCs) increases [Ca²⁺]_i, thus triggering the myocyte contraction [3]. In this study we related the dynamics of [Ca²⁺]_i changes induced by selective P2X-R stimulation to corresponding changes in the cell membrane potential and the kinetics of P2X-R mediated cationic current (I_P2X), and analised the mechanisms of purinergic [Ca²⁺]_i mobilisation in RVSMCs at sub-cellular level. Experiments were conducted on single RVSMCs freshly isolated from arcuate and interlobular arteries dissected from rat kidney [4]. Changes of [Ca²⁺]_i in RVSMCs loaded with Fluo-4 were visualised using fast x-y confocal imaging. Electrical recordings were performed using perforated-patch technique. Data are presented as mean±S.E.M. RT-PCR analysis conducted on 500 RVSMCs collected under the microscope with glass micropipette confirmed the expression of genes encoding P2X₁-R, P2X₄-R, P2Y₂-R and P2Y₆-R. The purity of phenotype of SMCs collected for RT-PCR analysis was confirmed by expression of the genes encoding SMC marker (SM-MHC) but not the markers for fibroblasts and endothelial cells (CD34), neurons (PGP9.5) and pericytes (NG2). Selective stimulation of P2X-Rs with 10 µM α,β-methylene adenosine 5’-triphosphate (AMP-CPP) evoked [Ca²⁺]_i transient initiated by sub-plasmalemmal [Ca²⁺]_i upstroke (SPCU) [5]. Combination of confocal Ca²⁺ imaging with electrical recordings revealed that: (1) peak of both the AMP-CPP -induced action potential and I_P2X preceded the peak of [Ca²⁺]_i transient; (2) kinetics of I_P2X was consistent with predominant contribution of P2X₁-Rs; (3) the amplitude of the [Ca²⁺]_i transient detected under voltage-clamp (V_h=-60 mV) was reduced by about 50% in comparison to that observed under current-clamp. These observations suggest that both Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCCs) and Ca²⁺ release from intracellular stores also contribute to AMP-CPP -induced [Ca²⁺]_i mobilisation. This was confirmed with selective pharmacological agents. AMP-CPP -induced [Ca²⁺]_i transient was attenuated by 56.9±3.3% (n=26) following depletion of intracellular Ca²⁺ stores by 10-min incubation with 10 µM cyclopiazonic acid and by 34.0%±3.3% (n=14) following block of VGCCs with 5 µM nicardipine. The [Ca²⁺]_i transient remaining in the presence of both drugs had a peak amplitude 31.4%±4.3% of that in control (n=16) and resulted from direct Ca²⁺ entry though P2X-Rs.