An intranasally delivered, live attenuated, temperature sensitive (ts) respiratory syncytial virus vaccine candidate, rA2cp248/404/1,030DeltaSH, exhibits a low level of genetic instability in clinical studies, in contrast to the relatively high stability of two similar candidates, cpts248/404 and rA2cp248/404DeltaSH. The latter strains, containing two ts mutations (248ts and 404ts), are partially growth restricted at 37 degrees C, whereas, rA2cp248/404/1,030DeltaSH contains an additional ts mutation (1,030ts) that increases attenuation and partially restricts virus growth at 35 degrees C. Since the maximum human airway temperature is 35.5 degrees C, we investigated whether growth restriction at 35 degrees C contributes to genetic instability of rA2cp248/404/1,030DeltaSH in vitro. We conducted in vitro passage studies with the three strains at 32 degrees C (a fully permissive growth temperature) and 35 degrees C (restrictive for only rA2cp248/404/1,030DeltaSH). Instability of the ts phenotype was observed only in rA2cp248/404/1,030DeltaSH passaged at 35 degrees C, and corresponded with reversion at the 248ts or 1,030ts mutation sites, as observed in clinical studies. This study indicates that ts mutations that partially restrict replication at physiologic temperatures may contribute to genetic instability of viruses in vivo. In vitro passage studies performed at appropriate temperatures can be used to assess genetic stability and to prioritize ts vaccine candidates for clinical evaluation.