To study the contribution of the Na+-Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]c were evoked by either depolarisation (−70 mV to 0 mV) or by release from the SR by caffeine (10 mm) or flash photolysis of caged InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+-induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation-evoked rise in [Ca2+]c. However, although Ca2+ was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation-evoked Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+-Ca2+ exchanger activity. Thus inhibition of the Na+-Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+-Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+-Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+-Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.