Reverse transcription-PCR (RT-PCR) is a sensitive method for detection of RNA virus nucleic acid sequences in clinical respiratory specimens. Previous studies have focused on RT-PCR for a single virus, but this approach is limited by the inability to establish a specific etiology when the RT-PCR result is negative and by the inability to document simultaneous infections involving more than one virus. The purpose of this study was to apply a panel of RT-PCR protocols for respiratory syncytial virus, parainfluenza virus, and picornaviruses to respiratory specimens from 80 children suspected to have acute viral respiratory tract infections and to correlate RT-PCR results with viral culture results and clinical diagnosis. In comparison with viral culture, the RT-PCR panel had a sensitivity of over 94% and showed evidence of simultaneous infections in a significantly greater proportion of specimens (20.0% versus 3.8%; P < 0.002). For specimens in which no viruses were detected by culture, the proportion of specimens with positive picornavirus RT-PCR results was significantly greater than the proportion of specimens with positive respiratory syncytial virus or parainfluenza virus RT-PCR results (P < 0.001). There were no statistically significant associations between RT-PCR results and clinical diagnosis. In summary, the RT-PCR panel provides an improved approach to obtain new insights into acute viral respiratory tract infections in children.