Cold-passaged (CP) mutants derived from the JS strain of wild type wt parainfluenza type 3 virus (PIV3) are being evaluated as candidate live virus vaccines. The wt virus was serially passaged 45 times at low temperature and mutant clones with the cold-adapted (CA), temperature-sensitive (ts), and attenuation (ATT) phenotypes were selected following passage levels 12, 18 and 45 (cp12, cp18, and cp45). The cp45 virus was more ts than the cp12 or cp18 mutants, although all 3 mutant viruses were clearly attenuated in rhesus monkeys compared to wild type virus. The mean peak titers of the cp12 and cp18 viruses administered by the intratracheal route were at least 6000-fold lower than JSwt in both the upper and lower respiratory tracts. The cp45 virus was not recovered from monkeys administered virus by the i.t. route alone; however, when the cp45 virus was administered by the intranasal route, it replicated in the upper respiratory tract to a level comparable to that of the cp12 and cp18 viruses, but continued to be markedly restricted in the lower respiratory tract. These data indicate that the cp12 and cp18 viruses contain predominantly non-ts attenuating mutations whereas the cp45 mutant has both non-ts and ts attenuating mutations. Each of the CP mutants induced a high level of resistance to wild type virus challenge. Also, the ATT phenotype of the cp12 and cp18 viruses as measured in rhesus monkeys was stable after replication in chimpanzees or humans, respectively, although the ts phenotype was not. Based on its greater level of temperature sensitivity in vitro and its greater degree of attenuation in rhesus monkeys, the cp45 virus appears to be the most promising vaccine candidate for humans.