Abstract: Several research studies involving fused heteroaromatic dyes based on the oxazine moiety have been focused on their absorption and fluorescence spectroscopic properties.¹ These long-wavelength fluorophores find applications as biological probes, namely in covalent labeling of amino acids, proteins, peptides and DNA.² Also, more frequently they have been used in non-covalent labeling, such as staining nucleic acids in a variety of context, monitoring protein conformation alterations or for therapeutic purposes.³ Although the interest of photophysical properties, several compounds possessing the phenoxazine nucleus have been stressed due to their antiproliferative properties with potential applications both as antitumour and as antimicrobial agents. Considering our promising previous results in this field,⁴ and having in mind future evaluation of biological activity, we decided to synthesise new benzo[a]phenoxazinium salts having chlorinated terminals at their substituents in 2- and 9- positions. Furthermore, these compounds can also be used as covalent and non-covalent probes, and consequently the photophysical properties were also studied in ethanol and water in simulated physiological conditions. Acknowledgements: Thanks are due to the Foundation for Science and Technology (Portugal) for its financial support of Centre of Chemistry. The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network (RNRMN) and was purchased in the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT. References 1. Jose, J.; Burgess, K. Tetrahedron 2006, 62, 11021-11037. 2. Salomi, B. S. B.; Mitra, C. K.; Gorton, L. Synth. Met. 2005, 155, 426-429. 3. Nakanishi, J.; Nakajima, T.; Sato, M.; Ozawa, T.; Tohda, K.; Umezawa, Y. Anal. Chem. 2001, 73, 2920-2928. 4. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Bioorg. Med. Chem. 2008, 16, 3274-3282.