We used human milk protein without casein (HMP) as a multi-protein preparation for studies of oral tolerance. The HMP mainly contained secretory IgA (S-IgA), lactoferrin and α-lactalbumin. Mice were given 1 mg or 25 mg of HMP by the oral route three times at weekly intervals, or 25 mg in 4 oral doses at weekly intervals prior to systemic immunization. It is interesting to note that antigen-specific serum IgG (IgG1>IgG2b) responses to HMP were induced by subsequent intravenous immunization with 100 μg of HMP at all doses of HMP given orally. These findings suggested that HMP contains multiple proteins which cannot induce mucosal (oral) tolerance. To further investigate possible molecular and cellular mechanisms which account for the absence of mucosal mediated tolerance following oral feeding with HMP, two groups of mice were given HMP by oral followed by the systemic route. Strong serum IgG1 and IgG2b responses to HMP were induced by oral immunization with 1 mg of HMP as well as intravenous immunization with 100 μg of HMP. Purified CD4+ T cells isolated from spleens of both orally and intravenously immunized mice produced an array of Th2 type cytokines including IL-4, IL-5, IL-6 and IL-10, but not IFN-γ and IL-2. These results suggest that both oral and intravenous administration of HMP induce exclusive Th2 type immune responses, which may prevent the development of mucosally induced tolerance.