We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca²⁺ spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01±0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca²⁺ spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca²⁺ spark rate (to 130.7±6.4%) within 5 seconds, followed by a return to near background levels (to 104.4±5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 µmol/L GsMTx-4), perfusion with Na⁺/Ca²⁺-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca²⁺ spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 µmol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular–sarcoplasmic reticulum complex (to within 10^-8m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca²⁺ spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation.