Rotaviruses are the single most important causes of severe acute diarrhoea in children worldwide. Despite success in developing vaccines, there is still a lack of knowledge about many components of the immune response, particularly those to non-structural proteins. This study established radioimmunoprecipitation (RIP) assays using labeled G1P[8], G2P[4], and G4P[6] human rotaviruses to examine the spectrum and duration of rotavirus antibodies in sera collected sequentially for 18-36 months from 27 children after hospitalization for primary rotavirus gastroenteritis. Five children experienced rotavirus re-infections. Primary responses detected to non-structural protein NSP2 declined to baseline after 100-150 days. Responses were heterotypic between NSP2 of G1P[8] and G4P[8] rotaviruses. Re-infections after 465-786 days boosted antibody levels to NSP2of both serotypes, together with the appearance of anti-NSP2 to G2P[4], even though there was no evidence of infection with this serotype. We developed an enzyme-immunoassay to measure sequential levels of anti-NSP2 IgG and IgA, using recombinant (heterotypic) NSP2 derived from SA11 (G3P[2]). Anti-NSP2 IgG and IgA were detected in sera from 23/23 (100%) and 18/24 (75%) of children after primary infection, declined to baseline after 100-150 days, were boosted after rotavirus re-infections, and again declined to baseline 150 days later. Anti-NSP2 IgA was also detected after primary infection, in duodenal juice from 14/16 (87%), and faecal extract from 11/19 (57%) of children. Sequential estimation of anti-NSP2 EIA levels in sera could be a sensitive index of rotavirus infection and re-infection. The potential of anti-NSP2 to limit viral replication after re-infection deserves further study.