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Prior studies, including one from our institution performed in 2001, suggest that nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) occurs infrequently in the healthy pediatric population (0.2–2.2%). However, infections caused by community-associated MRSA have increased remarkably in recent years. As a result, we restudied the prevalence of MRSA nasal colonization in healthy children, comparing results from 2001 and 2004.Nasal swabs were collected from 500 children presenting for health maintenance visits. Samples were cultured quantitatively, and MRSA isolates were confirmed by growth on selective media, coagulase testing and the presence of the mecA resistance gene. MRSA isolates were further analyzed for antibiotic susceptibilities, genetic relatedness by pulsed field gel electrophoresis and polymerase chain reaction for the detection of the gene encoding Panton-Valentine leukocidin.There were 182 children (36.4%) colonized with S. aureus, and 46 (9.2%) colonized with MRSA. This is significantly higher than the MRSA colonization rate in 2001 (0.8%; P < 0.001). There were no significant associations between potential risk factors and MRSA colonization except for having a family member work in a hospital (odds ratio, 2.0; 95% confidence interval, 1.03–4.1). Pulsed field gel electrophoresis revealed heterogeneity of circulating strains, and the Panton-Valentine leukocidin gene locus was detected in 10 of 46 MRSA isolates (22%).Nasal MRSA colonization in healthy children in Nashville has increased significantly in the past 3 years. As colonization typically precedes infection, this increase may be a major factor in the emergence of community-associated MRSA as a pathogen of healthy children.