For the first time in Uruguay the frequency of moderate to heavy colonization of the upper respiratory tract by Streptococcus pneumoniae was investigated in children with acute respiratory infections (n = 717) and in healthy controls (n = 564). Serotypes of S. pneumoniae were identified as colonizing and invasive strains and their susceptibility to antibiotics was determined. Semiquantitative cultures of nasopharyngeal aspirates yielded 42.1% of positives in ambulatory patients and 15.2% in controls. Throat swabs from hospitalized children and matched controls revealed, respectively, 18.4 and 11.5% colonization. Different sampling and culture procedures were evaluated. Seasonal variations in colonization were also detected. Geographic variations in serotype frequency and distribution were assessed. Serotype 14 was predominant among invasive and colonizing strains in patients. Ten serotypes, included in the 23-valent S. pneumoniae vaccine, were not detected. Intermediate resistance to penicillin was seldom observed, but 37.2% of the invasive strains were resistant to cotrimoxazole, indicating the need for a permanent surveillance of isolates to antibiotic susceptibility.