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Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenylalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl-tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant a subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. [ABSTRACT FROM AUTHOR]