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These last years, combination of high spatiotemporal resolution techniques with the development of fluorescent voltage sensitive dyes (VSDs) have significantly improved our understandings of electrophysiological functions of electrically excitable organs (heart, brain). These dyes allow to measure simultaneously membrane potential changes from multiple sites of biological preparations in a non-invasive way. However, conventional dyes have excitation spectra in the blue/green range, which limits the depth of penetration due to high absorbance and photon scattering properties. Therefore, the development of longer wavelength VSDs could have many advantages namely: action potentials recording from tissue's deeper layers, enhanced voltage sensitivity, and improved spectral properties such that simultaneous monitoring of multiple parameters (i.e. ion transient, pH).The goal of our study is to develop and characterize novel VSDs with large Stoke shifts and near-infrared spectral properties. Three families of VSDs were thus synthesized from two heterocyclic fluorophores and their properties (spectral properties, membrane staining, and dye phototoxicity) assessed in isolated ventricular cells and in Langendorff perfused rat hearts. In addition, special attention is paid to Signal:background ratios and signal kinetics of these new dyes by epifluorescence imaging.Optimal peak emission wavelengths for all dyes was found between 700 nm and 750 nm. The largest observed Stoke shift was found with fluorophore 1 family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore 2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye, Di-4-ANEPPS.Rapid signal decay indicative of internalization was observed for one VSD family. However, in other two, signal stability was improved compared to Di-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltage sensitivity and potential for applications.