Group A human rotavirus strains D, Wa, DS-1, and P were originally recovered from children with diarrhea. In an attempt to attenuate virulent, wild-type human rotaviruses of major epidemiological importance for use in a live oral vaccine, two reference rotavirus strains, D and DS-1, and two laboratory-generated reassortants, Wa x DS-1 and Wa x P, were subjected to cold adaptation. Collectively, these viruses provide antigenic coverage for both of the clinically important rotavirus VP4 antigens and three of the four important rotavirus VP7 antigens. Mutants of each of these rotaviruses were selected during successive serial passage in primary African green monkey kidney cells at progressively lower suboptimal temperatures (30, 28, and 26 degrees C). The genotype of each mutant appeared to be indistinguishable from that of its wild-type, parental virus. The mutants recovered after 10 serial passages at 30 degrees C exhibited both temperature sensitivity of plaque formation (i.e., a ts phenotype) and the ability to form plaques efficiently at suboptimal temperature (i.e., a cold adaptation [ca] phenotype), in contrast to parental wild-type rotavirus. The succeeding set of 10 serial passages at 28 degrees C selected mutants that exhibited an increased degree of cold adaptation, and three of the mutants exhibited an associated increase in temperature sensitivity. Finally, in the case of three of the strains, the third successive serial passage series, which was performed at 26 degrees C, selected for mutants with an even greater degree of cold adaptation than the previous series and was associated with greater temperature sensitivity in one instance. It appeared that each of the viruses sustained a minimum of four to five mutations during the total selection procedure. The ultimate identification of candidate vaccine viruses that exhibit the desired level of attenuation, immunogenicity, and protective efficacy needed for immunoprophylaxis will require evaluation of these mutants in susceptible humans.