The bright fluorescent cytosine analogue tC(O) stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tC(O), and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes ( = 0.22) that is virtually unaffected by the neighbouring bases (Φ(F) = 0.20-0.25), resulting in an average brightness of 1900 M(-1) cm(-1). The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield ( = 0.24) compared to dsRNA, with a broader distribution (Φ(F) = 0.17-0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tC(O)-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA ( = + 2.3 °C). These properties make tC(O) a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.