The local and systemic immune response to rotavirus was investigated, both in previously exposed adult sheep and gnotobiotic lambs. Vaccines composed of rotavirus or recombinant VP6 mixed with or incorporated into ISCOMs were used to examine if one oral dose could induce a mucosal immune response and protection against subsequent challenge. Gnotobiotic lambs were vaccinated orally either with PBS/ISC, inactivated rotavirus (IRV)/ISC, recombinant VP6/ISC, or IRV alone, challenged 3 weeks later with a live virulent lamb strain, and killed 8-9 days after challenge. The immune response was measured as described above. The rotavirus-vaccinated groups had RV IgG ASC and antibodies in blood from 7 and 11 days respectively. After challenge, rotavirus-vaccinated groups cleared the virus in a reduced period (6-7 days vs 8-9 days), however this was only significant (p<0.05) in the IRV/ISC group. RV IgA antibodies were observed in serum and nasal secretions from 4 days after challenge. In intestinal scrapings, these were significantly (p<0.05) higher in the IRV/ISC and IRV groups. RV IgG antibodies were significantly (p<0.05) increased in nasal secretions and intestinal scrapings in the IRV/ISC and IRV groups. Lymphocytes proliferated significantly in JPPs against rotavirus in the IRV/ISC and IRV groups. CD45R+ cells were significantly increased in blood in the IRV/ISC group before challenge, however no differences were found in other lymphocyte subpopulations either in blood or GALT. A downregulation of IFNγ transcripts was observed in JPPs and MLNs in the IRV/ISC group while the VP6/ISC group had a higher expression compared with the PBS/ISC and IRV groups. IL-4 transcripts were low in MLNs in all groups but in JPPs the rotavirus-vaccinated groups had a higher expression. IL-6 transcripts in JPPs were higher in the IRV/ISC and VP6/ISC groups but in MLNs all rotavirus-vaccinated groups had a higher level of expression. One oral dose of inactivated rotavirus alone, mixed with ISCOMs, or recombinant VP6 incorporated into ISCOMs, can induce priming and partial protection. These results suggest also that different immunological mechanisms take place when different vaccination protocols are used.