Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme. Three classes of mutations were found. (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; one insertion in particular was studied. This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNA structure were found. One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight. This protein is less stable than the wild-type enzyme. These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated.