Respiratory syncytial virus (RSV) is an important childhood pathogen of acute lower respiratory infections in developed and developing countries. The molecular epidemiology of RSV in India is largely unknown. The present study was undertaken to standardize and evaluate reverse transcription-PCR (RT-PCR) for the rapid and simultaneous detection of RSV groups A and B in clinical samples and to study intragroup genetic variability. RT-PCR was evaluated by comparing the results of seminested RT-PCR with centrifugation-enhanced cultures on 200 nasopharyngeal aspirates from children with acute lower respiratory infections. RSV was isolated in 34 nasopharyngeal aspirates by centrifugation-enhanced cultures and identified in 45 samples by RT-PCR. In 15 samples RSV was identified by seminested RT-PCR alone and in four by centrifugation-enhanced cultures alone. Of the 45 samples positive for RSV by nested PCR, 15 belonged to group A, 29 to group B, and one sample suggested a mixed infection. Group B RSV predominated in both years of the 2-year study. Genetic variability within RSV groups was studied by restriction fragment analysis of 35 PCR products. Among both group A and group B RSV, two different composite patterns were observed. Thus, RSV was found to be a major pathogen of acute lower respiratory tract infections in India, as it was detected in 24.5% of children by RT-PCR. RT-PCR provides a sensitive method for detection and typing of RSV group A and B viruses in clinical samples as well as a means to study intragroup variations. However, a higher sensitivity of detection of RSV in clinical samples can be obtained by its combination with additional techniques, such as virus cultivation.