It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca2+ transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca2+ transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca2+ measurement optics with a force measurement system using carbon fibers to examine the relation between Ca2+ transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca2+ loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca2+ transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca2+ transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca2+ transients reflect the affinity of troponin C for Ca2+, which is influenced by the change in strain on the thin filament but not by the length change per se.