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, G2P, and G4P 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 and G4P rotaviruses. Re-infections after 465-786 days boosted antibody levels to NSP2of both serotypes, together with the appearance of anti-NSP2 to G2P, even though there was no evidence of infection with this serotype. We developed an enzymeimmunoassay to measure sequential levels of anti-NSP2 IgG and IgA, using recombinant (heterotypic) NSP2 derived from SA11 (G3P). 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 reinfection deserves further study. J. Med. Virol. 80:1090-1098, 2008. © 2008 Wiley-Liss, Inc.