In the mammalian heart, the right ventricle (RV) has a distinct structural and electrophysiological profile compared to the left ventricle (LV). However, the possibility that myocytes from the RV and LV have different contractile properties has not been established. In this study, sarcomere shortening, [Ca²⁺]_i transients and Ca²⁺ and K⁺ currents in unloaded myocytes isolated from the RV, LV epicardium (LVepi) and LV endocardium (LVendo) of adult mice were evaluated. Maximum sarcomere shortening elicited by field stimulation was graded in the order: LVendo > LVepi > RV. Systolic [Ca²⁺]_i was higher in LVendo myocytes than in RV myocytes. Voltage‐clamp experiments in which action potential (AP) waveforms from RV and LVendo were used as the command signal, demonstrated that total Ca²⁺ influx and myocyte shortening were larger in response to the LVendo AP, independent of myocyte subtypes. Evaluation of possible regional differences in myocyte Ca²⁺ handling was based on: (i) the current–voltage relation of the Ca²⁺ current; (ii) sarcoplasmic reticulum Ca²⁺ uptake; and (iii) mRNA expression of important components of the Ca²⁺ handling system. None of these were significantly different between RV and LVendo. In contrast, the Ca²⁺‐independent K⁺ current, which modulates AP repolarization, was significantly different between RV, LVepi and LVendo. These results suggest that these differences in K⁺ currents can alter AP duration and modulate the [Ca²⁺]_i transient and corresponding contraction. In summary, these findings provide an initial description of regional differences in excitation–contraction coupling in the adult mouse heart. Evidence that the AP waveform is an important causative factor for these differences is presented.