The low prevalence of ciprofloxacin-resistant (Cp r) Streptococcus pneumoniae isolates carrying recombinant topoisomerase IV genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal parE-parC intergenic regions. A study of the transcription of these genes and of the fitness cost for 24 isogenic Cp r strains was performed. Six first-level transformants were obtained either with PCR products containing the parC quinolone resistance-determining regions (QRDRs) of S. pneumoniae Cp r mutants with point mutations or with a PCR product that includes parE-QRDR-ant-parC-QRDR from a Cp r Streptococcus mitis isolate. The latter yielded two strains, T6 and T11, carrying parC-QRDR and parE-QRDR-ant-parC-QRDR, respectively. These first-level transformants were used as recipients in further transformations with the gyrA-QRDR PCR products to obtain 18 second-level transformants. In addition, strain Tr7 (which contains the GyrA E85K change) was used. Reverse transcription-PCR experiments showed that parE and parC were cotranscribed in R6, T6, and T11; and a single promoter located upstream of parE was identified in R6 by primer extension. The fitness of the transformants was estimated by pairwise competition with R6 in both one-cycle and two-cycle experiments. In the one-cycle experiments, most strains carrying the GyrA E85K change showed a fitness cost; the exception was recombinant T14. In the two-cycle experiments, a fitness cost was observed in most first-level transformants carrying the ParC changes S79F, S79Y, and D83Y and the GyrA E85K change; the exceptions were recombinants T6 and T11. The results suggest that there is no impediment due to a fitness cost for the spread of recombinant Cp r S. pneumoniae isolates, since some recombinants (T6, T11, and T14) exhibited an ability to compensate for the cost.