In Europe, Streptococcus suis serotypes 2 and 9 are very important causative agents of meningitis, arthritis and septicemia in piglets. So far, no vaccine has been described which elicits protection against both serotypes. The working hypothesis of this study was that an isogenic serotype 2 mutant attenuated in virulence but not in colonization of the respiratory tract might induce protective immunity against both serotypes. Piglets were immunized with the attenuated S. suis serotype 2 strain 10Deltaofs2/12, which is deficient in expression of the serum opacity factor OFS. Three weeks after intranasal application of 10Deltaofs2/12 piglets were challenged intravenously with two strains representing the most important S. suis pathotypes in Europe, the homologous MRP+ EF+ SLY+ serotype 2 strain and a heterologous MRP* SLY+ serotype 9 strain. Application of the live vaccine elicited significant serum IgG responses against muramidase-released protein (MRP), extracellular factor (EF) and, most prominently, suilysin. Seroconversion against suilysin was accompanied with an increase of suilysin-neutralization titers in the vaccinated group. Though mortality was lower in the vaccinated groups, significant protection was not observed, neither against the homologous nor against the heterologous challenge. This was in agreement with the finding that the vaccinated piglets had low opsonizing antibody titers insufficient to mediate elimination of the two challenge strains by porcine neutrophils. In conclusion, a single intranasal application of the S. suis serotype 2 strain 10Deltaofs2/12 elicited humoral immune responses against different S. suis antigens but failed to induce sufficient opsonizing antibody titers and protective immunity against systemic serotypes 2 and 9 challenges.