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To determine the prevalence of penicillin-nonsuceptible Streptococcus pneumoniae (NS-SP) at 12 child-care centers (CCC) in urban and rural Nebraska and to evaluate the genetic diversity of pneumococcal strains present in the CCC environment.Nasopharyngeal cultures for S. pneumoniae were obtained from children 2 to 24 months old. Capsular serotyping, pulsed field gel electrophoresis (PFGE) and microbroth dilution MICs were performed for all S. pneumoniae. Antibiotic exposure was also evaluated as a potential risk factor for colonization with NS-SP.Nasopharyngeal colonization with S. pneumoniae was present in 121 (56%) of 215 children. The MICs of penicillin were 0.12 to 1.0 μg/ml for 57 (47%) and >1.0 μg/ml for 10 (8%) isolates. Clindamycin MICs of >0.5 μg/ml were found in 6 isolates (5%). MICs of ceftriaxone were 0.5 μg/ml in 28% of S. pneumoniae and 1.0 μg/ml in 7%. PFGE and capsular serotyping demonstrated multiple strains that were penicillin-nonsusceptible in both the urban and rural CCC. PFGE and capsular serotype defined shared strains within each CCC, but some PFGE “types” could be found in multiple serotypes. Antibiotic exposure during the 2 months before nasopharyngeal culture was not a statistically significant risk factor for nasopharyngeal colonization with NS-SP.NS-SP are highly prevalent in urban and rural Nebraska. PFGE similarities between serotypes may reflect “serotype switching” but may also reflect genetic similarity between S. pneumoniae strains.