Mutants of human rhinovirus serotype 14 (HRV14) with increased resistance to treatment at low pH were obtained by repeated cycles of exposure to pH 4.5 and propagation in HeLa cells. Whereas wild-type virus lost more than 5 logs of infectivity upon incubation at pH 4.3, the three isolates examined were essentially unaffected. Conformational change of the viral capsid upon exposure to low pH was assessed as an increase of hydrophobicity by partition between an aqueous phase and a Triton X-114 phase; the mutants required exposure to a much lower pH to accumulate in the Triton phase than wild-type HRV14. The sequence of the capsid region was determined for three isolates; two isolates were found to have the changes Thr17 to lie in VP2 and Asn 100 to lie in VP1. The third isolate also had the change Thr17 to Ile in VP2; however, in VP1, Asp101 was replaced by Glu. Separate introduction of the mutations into full length cDNA clones of the wild-type sequence of HRV14 showed that only the changes in VP1 were necessary for the increased stability at pH 4.5. The implications of the mutations for the three-dimensional structure of the viral capsid are discussed.