When myocardium is held in a stretched position, its [Ca2+]i and twitch force slowly increase over several minutes (slow force response to stretch: SFR). We have reported that TRPC3, non-selective cation channel, contributes to the SFR. Although their intracellular localization is important for the cellular function, it still remains unclear. To predict the localization of TRPC3, we investigated the changes in [Ca2+]SR during SFR in single isolated mouse cardiomyocytes in combination with mathematical model simulation study. Fura-4F-loaded cells were electrically stimulated at 1 Hz. A pair of carbon fibers was attached to cell ends to apply axial stretch. [Ca2+]SR was estimated by the caffeine-induced changes in [Ca2+]i before and after stretch (300 s). The stretch significantly increased the [Ca2+]SR, while C36I-A, TRPC3 blocker, diminished the increase in [Ca2+]SR. The results were reproduced only by the model with sarcolemmal stretch-activated cation channels, suggesting TRPC3 channels are located on sarcolemma.