Some bacterial and most organelle genomes do not encode the full set of 32 tRNA species required to read all codons according to Crick's wobble rules. 'Superwobble', in which a tRNA species with an unmodified U in the wobble position reads all four nucleotides in the third codon position, represents one possible mechanism for how a reduced tRNA set could still suffice. We have tested the superwobble hypothesis by producing knockout mutants for the pair of plastid glycine tRNA genes. Here we show that, whereas the tRNA gene with U in the wobble position is essential, the gene with G in this position is nonessential, demonstrating that the U-containing anticodon can indeed read all four glycine triplets. We also show that the price for superwobbling is a reduced translational efficiency, which explains why most organisms prefer pairs of isoaccepting tRNAs over the superwobbling mechanism.