The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.