The effect of introducing D-amino acid residues in an hexapeptide was examined both at the antigenic and immunogenic levels. A series of D-analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were produced. Four analogues contained a single change of an L-residue by the corresponding enantiomer, one peptide contained two D-residues and another one contained only D-residues (D-enantiomer). A peptide analogue was also synthesized in which the 2 Arg residues were replaced by Lys residues. The parent peptide and peptide analogues were injected into mice after covalent coupling to small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The substitution of L-Arg131 to Lys or D-Arg was found to change neither the antigenic nor immunogenic properties of the resulting peptides. In contrast, the substitution of Glu133, Arg134, and Ala135 by the respective enantiomers drastically altered the antigenicity of the modified peptides. Each of the six D-analogues induced an immune response with an unusually high level of IgG3 antibodies. The D-enantiomer produced IgG3 antibodies which reacted with the homologous peptide as well as with the all L-peptide and the parent protein H3 in solution but not with analogues containing one or two D-residues only. IgG3 antibodies produced against the all L-peptide reacted with the free all D-peptide but not with the other analogues containing D-residues in position 133, 134, and 135.