Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated.