Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet codon, rather than quadruplet codon, genetic code? Here, we investigate the tolerance of the genetic code to tRNA mutations that increase codon size. We found that tRNAs from all 20 canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.