In many types of smooth muscle cells, the sarcoplasmic reticulum (SR) has both peripheral and central components. Presumably, peripheral SR hinders calcium diffusion into cytosol during depolarisation induced calcium entry and contributes to calcium removal from cytosol (superficial buffer barrier, see reference for review). The superficial buffer barrier hypothesis implies that there should be gradients of cytosolic calcium concentration ([Ca²⁺]_i) between the cell periphery and the bulk of cytosol during depolarisation induced Ca entry. In this study we have investigated the spatio-temporal profile of depolarisation induced [Ca²⁺]_i transients in isolated uterine myocytes and its relation to the distribution of the SR within the cell. Late pregnant (19-21 days) Sprague-Dawley rats were killed by cervical dislocation after CO₂ anaesthesia, in accordance with Schedule 1 of the UK Home Office. Single cells were isolated from the longitudinal layer of the myometrium and superfused with Krebs solution. Patch clamp technique combined with Ca imaging was used to measure Ca current and [Ca²⁺]_i in different parts of the cell. Cells were loaded with the Ca sensitive dye Oregon Green 488 BAPTA (OG) via patch electrode. Images were acquired at video rate. Distribution of SERCA pump and ryanodine receptors in uterine smooth muscle cells were examined on a confocal microscope using fluorescently labelled thapsigargin and ryanodine. The surface membrane was visualised using membrane-associated dyes di-8-ANEPPS and C18-Calcium Green. Depolarisation of the cell membrane from a holding potential of -60 mV to 0 mV elicited inward calcium current (540±72 pA, n=7) accompanied by [Ca²⁺]_i transient. [Ca²⁺]_i rose synchronously throughout the entire cell. The amplitude of [Ca²⁺]_i transients measured as normalised OG signal was higher in the middle of the cell compared to the cell periphery. Confocal microscopy of the di-8-ANEPPS or C18-Calcium Green stained cells revealed multiple invaginations of the surface membrane similar to the T-tubules in striated myocytes, although not as regular. SR was distributed both centrally and peripherally showing close apposition to the surface membrane invaginations preferentially on the cell periphery. Apparent synchronicity of the [Ca²⁺]_i transients and differences in their amplitude may be explained if L-type calcium channels are located within the membrane invaginations at higher density than on the surface of the cell.