Transfer of fluoroquinolone (FQ) resistance determinants between Streptococcus pneumoniae and viridans streptococci was explored by transformation in vitro. One-step FQ-resistant parC mutants were selected, and resistance could be transferred from DNA from S. oralis, S. mitis, S. sanguis, and S. constellatus to S. pneumoniae, with frequencies of 10(-3) to <10(-7) in correlation with the homologies of their quinolone resistance determining region sequences (95%, 91%, 85%, and 81%, respectively). Reciprocal transfers of mutated parC from DNA from S. pneumoniae to S. mitis and S. oralis were also observed. Simultaneous transfer of mutated parC and gyrA genes from S. mitis to S. pneumoniae yielded high-level-resistant pneumococcal transformants in one step at low frequencies. The parE-parC region of the type strain S. mitis 103335T had >90% homology with that of S. pneumoniae. The efficient interspecific transfer of quinolone resistance determinants in vitro leads us to anticipate their dissemination in the clinical setting.