The main goal of this study is to investigate the role of mitochondrial [Ca²⁺], [Ca²⁺]_m, in the possible up-regulation of the NADH production rate during increased workload. Such up-regulation is necessary to support increased flux through the electron transport chain and increased ATP synthesis rates. Intact cardiac trabeculae were loaded with Rhod-2(AM), and [Ca²⁺]_m and mitochondrial [NADH] ([NADH]_m) were simultaneously measured during increased pacing frequency. It was found that 53% of Rhod-2 was localized in mitochondria. Increased pacing frequency caused a fast, followed by a slow rise of the Rhod-2 signal, which could be attributed to an abrupt increase in resting cytosolic [Ca²⁺], and a more gradual rise of [Ca²⁺]_m, respectively. When the pacing frequency was increased from 0.25 to 2Hz, the slow Rhod-2 component and the NADH signal increased by 18 and 11%, respectively. Based on a new calibration method, the 18% increase of the Rhod-2 signal was calculated to correspond to a 43% increase of [Ca²⁺]_m. There was also a close temporal relationship between the rise (time constant ∼25s) and fall (time constant ∼65s) of [Ca²⁺]_m and [NADH]_m when the pacing frequency was increased and decreased, respectively, suggesting that increased workload and [Ca²⁺]_c cause increased [Ca²⁺]_m and consequently up-regulation of the NADH production rate.