Rapid carbohydrate antigen tests are frequently used to diagnose group A streptococcal (GAS) pharyngitis. Despite evidence of modest sensitivity in medical settings, rapid antigen tests are available to the public for self-testing. We sought to determine if the personnel performing a rapid streptococcal antigen test influence the test's performance characteristics. Throat swabs of pediatric patients performed for GAS pharyngitis in a tertiary-care children's hospital network were included during two study periods in 2004 and 2005. The performance characteristics of a rapid carbohydrate antigen test were evaluated in three clinical settings against a nucleic acid probe test method according to the personnel performing the test (laboratory technologist versus nonlaboratory personnel). Between the study periods, nonlaboratory personnel from one site underwent retraining. Subsequently, the performance characteristics of the rapid antigen test were reassessed. The sensitivity of the rapid antigen test varied widely among the different testing sites (56 to 90%). Notably, test sensitivity was consistently greater when the test was performed by laboratory technologists than when it was performed by nonlaboratory personnel (P < 0.0001). Although the rapid antigen test sensitivity significantly improved after nonlaboratory personnel at one testing site were retrained (sensitivity before versus after retraining; P < 0.0001), the sensitivity remained greater in the laboratory technologist cohort (P < 0.0001). These data confirm the important relationship of the operator performing a rapid streptococcal antigen test with the test's accuracy, even in a clinical setting, where operator training is mandated. Therefore, its use outside the medical setting by lay persons cannot be recommended without culture backup.