Abstract: The use of fluorescent probes is a well-established practice in many scientific and technologic areas with several decades. Nevertheless, developing new fluorophores is fundamental to overcome the limitations associated with the commercially available fluorescent probes. The potential as fluorescent probes of two recently synthesised water-soluble benzo[a]phenoxazinium salts, mono- or dissubstituted with carboxylic ester groups as terminal of propyl substituents at the amine of position 9, were studied by fluorescence microscopy employing intact, live cells. With the aim of assessing the compounds cellular staining pattern and potential specificity, co-localization experiments were performed using yeast mutants expressing green fluorescent protein (GFP)-tagged proteins that localize to specific intracellular location/organelles. It was found that both compounds display a very high affinity to the vacuolar membrane, being the dissubstituted compound especially important due to its higher fluorescence at lower, less toxic concentrations.