Abstract: The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side-chain and sometimes an additional 2-thio (s(2)) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient yeast cells to test the wobble rules in vivo. We show that mcm(5) and ncm(5) side-chains promote decoding of G-ending codons and that concurrent mcm(5) and s(2) groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm(5)U34- and some ncm(5)U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.