Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children.Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 153:1. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r 2 ‫؍‬ 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 ؎ standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r 2 ‫؍‬ 0.158; P < 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P < 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in infants. In the United States, 3% of all previously healthy infants are hospitalized within their first year of life due to RSV infection (1), and this rate is increasing (15), making RSV the single most common cause of infant hospitalization (11). Although infants with chronic lung disease, congenital heart disease, or immunodeficiencies are at increased risk for severe RSV disease, most infants with severe RSV disease requiring hospitalization were previously healthy (1,18,19). Furthermore, RSV is also an increasingly recognized major cause of morbidity and mortality in debilitated adults and the elderly (17).Currently, our understanding of the pathogenesis of RSV in humans is incomplete. Relating viral load and viral dynamics to disease severity will expand our understanding of RSV pathogenesis. Studying RSV pathogenesis and evaluating potential vaccine and antiviral treatment strategies for humans requires quantitative tests for RSV. At present, the quantity of RSV obtained from clinical samples in such studies has been measured by quantitative culture using plaque assays. However, this method is time consuming and difficult to perform and has numerous inherent limita...