A sample of 28 penicillin-resistant Streptococcus pneumoniae strains isolated between 1991 and 1993 in a large hospital in Toulouse, France, was characterized by pulsed-field gel electrophoresis of genomic DNA. Also included were 6 penicillin-susceptible clinical isolates from Toulouse and 12 penicillin-resistant strains from different parts of the world. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field-inversion gel electrophoresis. Seven major pattern types could be recognized among the penicillin-resistant isolates from Toulouse. Nine of these isolates could be assigned to two clones that were also found in Spain and were associated with serotypes 6B and 9V. A third clone was isolated in South Africa and in Spain and contained serotype 23F isolates. The profiles obtained by field-inversion gel electrophoresis suggested that 15 of the 16 penicillin-resistant serogroup 23 isolates from Toulouse belonged to the same Spanish 23F clone. The molecular test profiles of penicillin-susceptible strains differed from those of resistant strains of the same serotype except those of 9V strains. These data underline the importance of the geographic spread of resistant clones from Spain in the emergence of penicillin-resistant pneumococci in France.