Antibody response in egg-laying hens was examined following repeated immunization with a human rotavirus vaccine. Four different rotavirus subtypes which have been isolated from fecal samples of infants in Korea were used in vaccine preparation. Following vaccination in hens at 0, 2, 4, and 8 weeks, neutralization antibody to rotavirus subtypes was first detected at 3 weeks and antibody titer was peaked at 8 weeks after the first vaccination. High antibody titers were maintained for up to 10 weeks after the first vaccination. The egg yolk antibody titers were stable with slight decrease (29.65%) by freeze-drying process. A high concentration (50 g/kg) of the yolk antibodies caused no clinical sign, body weight loss, and necropsy lesion following oral administration in mice. In addition, the yolk antibodies did not cause mortality for suckling mice when they were inoculated intracerebrally. Furthermore, yolk antibodies caused no cytopathic effects on two different cell lines. In conclusion, egg yolk containing high concentrations of human rotavirus antibodies can be produced by immunization in hens. The egg yolks appear to be safe for human consumption.