T-wave alternans, a powerful marker of arrhythmic events, results from alternation in action potential duration (APD). The underlying cellular mechanism of APD alternans is unknown but has been attributed to either intracellular calcium (Ca²⁺) cycling or membrane ionic currents, manifested by a steep slope of cellular APD restitution. To address these mechanisms, high-resolution optical mapping techniques were used to measure action potentials and Ca²⁺ transients simultaneously from hundreds of epicardial sites in the guinea pig model of pacing-induced T-wave alternans (n=7). The pacing rates (ie, alternans threshold) at which T-wave (369±11 bpm), APD (369±21 bpm), and Ca²⁺ (371±29 bpm) alternans first appeared were comparable. Importantly, the site of origin of APD alternans and Ca²⁺ alternans consistently occurred together near the base of the left ventricle, not where APD restitution was steepest. In addition, APD and Ca²⁺ alternans were remarkably similar both spatially and temporally during discordant alternans. In conclusion, the mechanism underlying T-wave alternans in the intact heart is more closely associated with intracellular Ca²⁺ cycling rather than APD restitution.