The novel fluorescent DNA cytosine base analogue 1,3-diaza-2-oxophenothiazine, tC, has previously been shown to have a remarkably preserved high quantum yield upon incorporation into a single strand of peptide nucleic acid, PNA, as well as when the latter is hybridized with a complementary DNA to form a PNA-DNA duplex. Here, we investigate fundamental photophysical properties of tC. Using fluorescence anisotropy, stretched film linear dichroism, and quantum chemical calculations, the transition moment polarizations of the lowest lying electronic states are determined. The neutral, base-pairing form of tC, having a fluorescence quantum yield of 0.2, is found to be the totally predominant species in a wide pH interval, 4-12. We show that the absorption band of tC at lowest energy, centered at 26 700 cm(-1) and well separated from the nucleobase absorption, is due to a single electronic transition polarized approximately at 35degrees from the long axis of the molecule. The 2-deoxyribonucleoside of 1,3-diaza-2-oxophenothiazine, synthesized for further incorporation into DNA, was found to display a fluorescence quantum yield nearly the same as in the form of tC that was incorporated into PNA, confirming the notion of the tC nucleoside being a probe with very promising fluorescence properties essentially invariant of environment, also upon incorporation into a DNA strand and upon hybridization.