Increases in contraction amplitude following rest or in elevated extracellular [Ca²⁺] have been attributed to increased sarcoplasmic reticulum (SR) Ca²⁺ stores and/or increased trigger Ca²⁺. However, either manipulation also may elevate diastolic [Ca²⁺]. The objective of this study was to determine whether elevation of diastolic [Ca²⁺] could contribute to positive inotropy in isolated ventricular myocytes. Voltage clamp experiments were conducted with high resistance microelectrodes in isolated myocytes at 37^oC. Intracellular free [Ca²⁺] was measured with fura-2, and cell shortening was measured with an edge detector. SR Ca²⁺ stores were assessed with 10 mM caffeine (0 mM Na⁺, 0 mM Ca²⁺). Following a period of rest, cells were activated with trains of pulses, which generated contractions of increasing amplitude, called positive staircases. Positive staircases were accompanied by increasing diastolic [Ca²⁺] but no change in Ca²⁺ transient amplitudes. When extracellular [Ca²⁺] was elevated from 2.0 to 5.0 mM, resting intracellular [Ca²⁺] increased and resting cell length decreased. Amplitudes of contractions and L-type Ca²⁺ current increased in elevated extracellular [Ca²⁺] although SR Ca²⁺ stores, assessed by rapid application of caffeine, did not increase. Although Ca²⁺ transient amplitude did not increase in 5.0 mM extracellular [Ca²⁺], diastolic [Ca²⁺] continued to increase with increasing extracellular [Ca²⁺]. These data suggest that increased diastolic [Ca²⁺] contributes to positive inotropy following rest or with increasing extracellular [Ca²⁺], in guinea pig ventricular myocytes.