Abstract: Five benzo[a]phenoxazinium dyes containing alkyl chains with twenty carbon atoms on 5- or 9-positions of the tetracyclic ring were efficiently synthesised and characterised by UV/Visible and fluorescence spectroscopy. The absorption and emission maxima in ethanol lie in the range 627-641 nm and 645-676 nm, respectively, with quantum yields varying from 0.14 to 0.38. Preliminary photophysical studies in zwitterionic (2,3-bis(palmitoyloxy)propyl 2- (trimethylammonio)ethyl phosphate, DPPC) and cationic (N,N-dimethyl-Noctadecyloctadecan- 1-aminium bromide, DODAB) vesicles are reported showing that these molecules are able to detect the gel to liquid-crystalline lipid phase transition through variations either in H-aggregation extent or in an acid-base equilibrium.