Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.