A confocal Ca²⁺ imaging technique was used to detect sub-cellular [Ca²⁺]_i changes induced by activation of P₂X purinoceptors (P₂X-Rs) with 10 µM α,β-methyleneadenosine 5-triphosphate (α,β-meATP) in smooth muscle cells (SMCs) freshly isolated from the guinea-pig vas deferens. The spatio-temporal patterns of the α,β-meATP – induced [Ca²⁺]_i changes detected with fast x-y confocal imaging in fluo-3 loaded SMCs were related to spatial distribution of the sarcoplasmic reticulum (SR), visualised with Brefeldin A BODIPY, and type 1 IP₃Rs and RyRs, visualised by indirect immunofluorescence method. We have found that sub-plasmalemmal (sub-PM) SR elements possess both type 1 IP₃Rs and RyRs, while central/perinuclear SR elements are enriched with RyRs. α,β-meATP -induced [Ca²⁺]_i mobilisation consisted of: (1) an initial sub-PM [Ca²⁺]_i upstroke (SPCU) followed by (2) [Ca²⁺]_i wave which propagated through the entire cell volume. The peak amplitude of the α,β-meATP -induced SPCU was reduced: (1) by 38±3% (n=3) after inhibition of RyRs (with 100 µM tetracaine); (2) by 48±4% (n=4) after inhibition of IP₃Rs (with 30 µM 2-APB) and (3) by 71±5% (n=3) following block of voltage-gated Ca²⁺ channels (VGCCs) (with 5 µM nicardipine). The cumulative inhibition of IP₃Rs, RyRs and VGCCs reduced the SPCU by 65±5% (n=6). Depletion of intracellular Ca²⁺ stores by 10 min incubation with 10 µM cyclopiazonic acid (CPA) decreased SCPU by 55±7% (n=8). Subsequent inhibition of VGCCs, while keeping the stores depleted, decreased SPCU by 63±4% (n=8). Recording of isometric tension of smooth muscle strips from the guinea-pig vas deferens revealed that stimulation of P₂X-Rs with α,β-meATP induced biphasic contraction. Inhibition of IP₃Rs with 30 µM 2-APB decreased the transient component of the contractile response by 60±5% (n=4) and completely removed the tonic component of the contraction. These results suggest that IP₃R-mediated Ca²⁺ release is involved in genesis of SPCU and contraction in response to P₂X-R activation, which is not linked to Gq/11- phospholipase C – IP₃ signalling pathway.