Nasopharyngeal carriage of potential pathogens was studied in 425 healthy 3- to 6-year-old children attending 16 day-care centres (DCCs) in nine Czech cities during the winter 2004–2005. The overall carriage of pathogens was 62·8% (Streptococcus pneumoniae, 38·1%; Haemophilus influenzae, 24·9%; Moraxella catarrhalis, 22·1%; Staphylococcus aureus, 16%). An age-related downward trend was observed for colonization with respiratory pathogens in contrast to Staph. aureus whose carriage was significantly higher among older children. The following serotypes of colonizing S. pneumoniae were the most predominant: 23F (20·6%), 6A (15·1%), 6B (12·7%), 18C (7·8%), 15B and 19F (6% each). The majority (94·3%) of H. influenzae isolates were non-typable; among capsulated isolates, serotype b was not found. Decreased susceptibility to penicillin was determined in 3% of pneumococci; 4·6% of H. influenzae strains and 85·1% of M. catarrhalis strains produced β-lactamase. As for non-β-lactam antibiotics, pneumococci resistant to trimethoprim–sulphamethoxazole were the most common (15·7%) among the attendees.