Microbiologic, serologic, and molecular typing techniques were used to characterize 272 isolates of Streptococcus pneumoniae colonizing or infecting children in Iasi, Romania, during a surveillance study conducted in 1996-1998. The 574 children in the study were from the following groups: healthy children attending 2 institutions, healthy children hospitalized for elective surgery, hospitalized children with pneumococcal infections, and human immunodeficiency virus (HIV)-infected children in an orphanage. Pneumococci colonizing healthy children from closed communities showed close similarities to pneumococci from children with pneumococcal infections; they expressed a limited number of similar serotypes, showed high frequency of penicillin and multidrug resistance, and shared several common clonal types. In contrast, isolates recovered from healthy children hospitalized for elective surgery expressed a large variety of serotypes, were less frequently resistant to antimicrobial agents, and showed great genetic diversity. Pneumococcal flora colonizing HIV-infected children showed a more complex epidemiology. These observations suggest a possible epidemiologic connection between the flora of S. pneumoniae colonizing healthy children in closed communities and the flora found in children hospitalized for infection.