Active efflux has been involved in fluoroquinolone resistance in Streptococcus pneumoniae. While only one efflux machinery has been well described (PmrA), the eventual existence of other pumps than PmrA has been suggested. The actual role of active quinolone efflux in producing ciprofloxacin resistance was examined by means of several methods. Strains HUB 2375 and HUB 3073 are clinical isolates resistant to fluoroquinolones. Since no mutations in the quinolone resistance-determining regions of these strains were found, resistance to fluoroquinolones should be attributed only to efflux. Strain R6 was transformed by DNA from both HUB 2375 and HUB 3073. Selected transformants were chosen for knockout of pmrA genes by polymerase chain reaction ligation mutagenesis in order to study the actual role and the degree of involvement of PmrA in determining ciprofloxacin resistance. Results strongly supported the hypothesis that efflux pumps other than PmrA are involved in fluoroquinolone resistance in clinical strains of pneumococci.