During sympathetic nerve activity ATP is co-released with noradrenaline leading to constriction in muscular arteries. Current views support the notion that physiological contractile responses to ATP are mainly mediated through activation of homomeric P2X1 receptors of the membrane of vascular smooth muscle cells (VSMCs), while the role of other P2X receptors (P2XRs) remains unclear. In this work we studied P2XRs in rat middle cerebral arteries (RMCA). RT-PCR gene expression analysis was performed using preparations from isolated VSMCs. The expression of proteins was confirmed by immunocytochemistry. Agonist-induced [Ca2+]i changes were monitored using x-y confocal imaging in fluo-3 loaded VSMCs. Electrical recordings were performed using patch-clamp technique. Isometric tension recordings were performed using wire myograph. Our results show that RMCA VSMCs express genes encoding only P2X1 and P2X4 receptor subunits and corresponding proteins in their plasma membrane. Both ATP and a potent P2XR agonist, α,β-meATP, evoked dose-dependent increase in [Ca2+]i of similar amplitude for each concentration with an EC50=0.38±0.29 µmol/l (n=7-17) and EC50=0.59±0.24 µmol/l (n=7-12) for ATP and α,β-meATP respectively. Sub-maximal concentration of noradrenaline (100 µmol/l) produced less than 10% of amplitude of [Ca2+]i increase evoked by 100 µmol/l ATP in RMCA VSMCs. Under current-clamp recordings 10 µmol/l ATP evoked either fast transient depolarisation or transient response followed by a sustained depolarisation or only sustained depolarisation of the membrane of different VSMCs. Similarly, under voltage-clamp both10 µmol/l ATP and 10 µmol/l α,β-meATP evoked either transient rapidly desensitising current (Ifp2x) or biphasic current consisting of transient and sustained component or only slowly desensitising current (Isp2x) in different VSMCs. Ifp2x rapidly desensitised within 3 s of α,β-meATP application, and completely recovered within 8 min after first application of the agonist. Selective P2XR blocker NF279 inhibited Ifp2x in dose dependent manner with an IC50=17±2 nmol/l (n=5-6) and complete inhibited the current at 10 µmol/l. NF279 inhibited Isp2x in dose dependent manner with IC50=25±6 µmol/l (n=4-5). These data suggest that monomeric P2X1R predominantly contribute to Ifp2x while heteromeric P2X1/4R predominantly contribute to Isp2x. Isometric tension recordings showed that both 10 µmol/l ATP and 10 µmol/l α,β-meATP produced biphasic contractions of RMCA preparations. Incubation of arterial segments with 10 µmol/l NF279 inhibited α,β-meATP (10 µmol/l) evoked contraction by 75±3% (n=6). 100 µmol/l noradrenaline produced only 8±2% (n=7) of contraction evoked by 10 µmol/l α,β-meATP. Our data suggest that in RMCA ATP may play role of the predominant neurotransmitter and that both homomeric P2X1R and heteromeric P2X1/4R contribute to ATP evoked constriction of these blood vessels.