The ribose 2′-hydroxyl group confers upon RNA many unique molecular properties. To better appreciate its contribution to structure and stability and to monitor how substitutions of the 2′ hydroxyl can alter an RNA molecule, each loop pyrimidine ribonucleotide in the UUCG tetraloop was substituted with a nucleotide containing either a fluorine (2′-F), hydrogen (2′-H), amino (2′-NH2), or methoxy (2′-OCH3) group, in the context of both the C:G and G:C loop-closing base pair. The thermodynamic parameters of these tetraloop variants have been determined and NMR experiments used to monitor the structural changes resulting from the substitutions. The modified riboses are better tolerated in the G[UUCG]C tetraloop, which may be due to its increased loop flexibility relative to the C[UUCG]G loop. Even for these simple substitutions, the free-energy change reflects a complex interplay of hydrogen bonding, solvation effects, and intrinsic pucker preferences of the nucleotides.