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We monitored the timing of acquisition of nasopharyngeal colonization ofStreptococcus pneumoniaein 125 healthy infants during their first 2 years of life.S. pneumoniaewas isolated at least once from 59 (47%) of 125 infants aged between 2 and 18 months. Twenty-four infants (19%) were colonized with penicillin-resistantS. pneumoniaeat some time during the study. During the course of this investigation, we identified sequential pneumococcal isolates of the same serotype from 5 infants, in which the penicillin minimum inhibitory concentration (MIC) increased over time. For 4 of the 5 infants, sequential isolates were identical, as determined by pulsed-field gel electrophoresis. SequentialS. pneumoniaenasopharyngeal isolates from some healthy infants demonstrated drift in penicillin MIC values over time, from penicillin-susceptible to penicillin-resistant.