Quantification of subcellularly resolved Ca²⁺ signals in cardiomyocytes is essential for understanding Ca²⁺ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca²⁺-dependent fluorescence changes into [Ca²⁺] changes. Therefore, we determined the in situ characteristics of a frequently used Ca²⁺ indicator, Fluo-4, and a ratiometric Ca²⁺ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca²⁺ signals in isolated cardiomyocytes. Ca²⁺ calibration curves revealed significant differences in the apparent Ca²⁺ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca²⁺]. Resting and diastolic [Ca²⁺] were always higher in the nucleoplasm. Systolic [Ca²⁺] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca²⁺] in the nucleoplasm. Ca²⁺ store depletion or blockade of Ca²⁺ leak pathways eliminated the resting [Ca²⁺] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca²⁺] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca²⁺] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca²⁺ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca²⁺ stores.