The inducible nonenzymatic chloramphenicol resistance (Cmr) determinant of the IncP plasmid R26 was cloned on a 1,900-base-pair restriction endonuclease HindIII fragment. Transposon Tn5 mutagenesis revealed that at least 1,400 base pairs is required for expression of Cmr. There was no increase in the level of Cmr when the copy number of the determinant was raised by cloning in pBR322 or pUB5572. Expression of Cmr by cells carrying a lower-copy-number pUB5572cml+ plasmid was inducible and thus indistinguishable from those with R26 itself. However, pBR322cml+-carrying cells expressed Cmr constitutively, possibly due to the activity of vector promoters or an elevated copy number. Transcriptional and translational cml-lac fusions were constructed. The operon (transcriptional) cml-lac fusion carried by the low-copy-number plasmid pUB5572 caused a low level of constitutive beta-galactosidase activity, which could not be elevated by induction with chloramphenicol and was not affected by a coresident R26cml+ element. In contrast, the gene (translational) cml-lac fusion expressed low-level beta-galactosidase activity, which was elevated fivefold by prior exposure to chloramphenicol. We conclude that the regulation of Cmr occurs posttranscriptionally.