One of the eight endonuclease EcoRI fragments of yeast DNA that hybridize to yeast tRNATyr has been identified with the genetically defined nonsense-suppressor locus SUP4. This identification was achieved by analyzing the meiotic linkage between the genetic determinant for the SUP4 phenotype and that for an electrophoretic variant of the EcoRI fragment. The SUP4 gene was then cloned from an ochre-suppressing yeast strain and analyzed by DNA sequencing. A wild-type SUP4 gene and two other genetically unidentified tRNATyr genes were also sequenced. The sequence of the ochre suppressor differs from that of the wild-type genes by virtue of a G.C leads to T.A transversion in the base pair that codes for the wobble position base of the tRNATyr anticodon. All four genes contain, immediately to the 3' side of the anticodon triplet, a 14 base pair tract that is not present in mature tRNATyr. Although the four genes, which represent three unlinked chromosomal loci, all encode the same mature tRNA sequence, there is virtually no observable sequence homology between the three loci in the region preceding the 5' end of the mature tRNATyr sequences.