Nasopharyngeal carriage of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was studied in 259 children attending day care centers (DCC) in Amsterdam, The Netherlands, and in 276 control children. The DCC children were sampled a second time after 4 weeks. Carriage rates for DCC children and controls were 58 and 37% for S. pneumoniae, 37 and 11% for H. influenzae, and 80 and 48% for M. catarrhalis, respectively. No increased antibiotic resistance rates were found in strains isolated from DCC children. All H. influenzae isolates were typed by random amplified polymorphic DNA (RAPD) analysis. Evidence for frequent transmission of H. influenzae strains within DCC was found. In the control group only two isolates (4%) displayed identical RAPD types versus 38% of strains from DCC children. Colonization with H. influenzae appeared to be short-lived in these children; more than half of the children harboring H. influenzae in the first sample were negative in the second sample, whereas most children still positive in the second sample had a different genotype than in the first sample. Of the newly acquired strains in the second sample, 40% were identical to a strain that had been found in a child in the same DCC in the first sample. DCC are to be considered epidemiological niches with a high potential for the spread of pathogenic microorganisms.