The aim of this study was to determine the factors favouring Streptococcus pneumoniae nasopharyngeal colonization of healthy children attending daycare centres and to describe the circulation of penicillin-nonsusceptible strains using molecular techniques. A single nasopharyngeal swab was obtained from 610 children attending daycare centres in the southeast area of Rome. Streptococcus pneumoniae isolates were serotyped, and antibiotic susceptibility was assayed by the E test. The genetic determinants of erythromycin resistance were detected by a duplex polymerase chain reaction, and the penicillin-nonsusceptible isolates were typed by pulsed-field gel electrophoresis. The overall carriage rate of Streptococcus pneumoniae was 14.9%. Living with more than three persons in the same household was the only risk factor statistically associated with carriage. Sixteen of 85 (18.8%) strains were nonsusceptible to penicillin, and 44 (52%) were resistant to erythromycin. Of the erythromycin-resistant strains, the vast majority showed a high level of resistance and carried the erm(B) gene. The penicillin-nonsusceptible strains belonged to six different serotypes; molecular typing showed that in only one case (2 strains) was there a circulation of the same clone in the same daycare centre. In view of the high rate of resistant Streptococcus pneumoniae strains, risk factors for carriage of resistant strains were evaluated. Children who received macrolides in the previous month had a higher risk of being colonized by macrolide-resistant strains as well as by strains resistant to both penicillin and erythromycin. Limiting the use of antibiotics in children seems the most appropriate measure to control the spread of antibiotic-resistant strains.