Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.