We obtained nasopharyngeal cultures for Streptococcus pneumoniae from 54 children ages 2 to 24 months attending an Omaha child-care center (CCC) in April 1994. Thirty-two (59%) of the 54 children were colonized with S. pneumoniae belonging to serotypes 23, 19, 6 and 11. Seventeen (53%) of the pneumococcal isolates were highly resistant to penicillin (minimal. inhibitory concentration greater than or equal to 2.0 mu g/ml; HR-SP) and 7 (22%) were intermediately resistant to penicillin (0.12 less than or equal to minimal inhibitory concentration less than or equal to 1.0 mu g/ml; IR-SP). Within each pneumococcal capsular serotype, there were 1 to 3 DNA subtypes based on pulsed field gel electrophoresis analysis. A single pulsed field gel electrophoresis strain predominated in most CCC rooms, suggesting horizontal transmission among cohorted children. Nasopharyngeal cultures obtained 4 months later revealed similar S. pneumoniae colonization rates (28 of 52, 54%); however, only 2 (7%) of 28 isolates were HR-SP and 11 (39%) were IR-SP, Colonization with resistant pneumococci persisted after 4 months in 4 (12%) of 34 children cultured on both occasions, Antibiotic use by attendees had decreased notably between the two sampling periods, suggesting that selective pressure within the CCC might contribute to seasonal variation in colonization rates with HR-SP and IR-SP, We conclude that multiple genetic clones of penicillin-resistant pneumococci call occur simultaneously in a single CCC, especially during periods of heavy antibiotic selection pressure, However, individual clones of penicillin-resistant S. pneumoniae may be spread from child to child, suggesting that colonization with penicillin-resistant S. pneumoniae should now be considered a CCC-associated phenomenon.