Proteus mirabilis PM13 is a well-characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50 micrograms ml-1) at a plating efficiency of 10(-4) to 10(-5). Such chloramphenicol-resistant colonies exhibit a novel phenotype with respect to chloramphenicol resistance. When a single colony grown on chloramphenicol agar is transferred to liquid medium and grown in the absence of antibiotic for 150 generations, a population of predominantly sensitive cells arises. This mutation-reversion phenomenon has been observed in other Proteus species and Providencia strains, wherein resistance has been shown to be mediated in each case by the enzyme chloramphenicol acetyltransferase. The cat gene responsible for the phenomenon is chromosomal and can be cloned from P. mirabilis PM13 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kilobase PstI fragment irrespective of the source of host DNA. The location of the cat gene within the PstI fragment was determined by Southern blotting with a cat consensus oligonucleotide corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase, and the direction of transcription was deduced from homology with the type I cat variant.