Heat shock proteins (HSP) like Hsp70 and gp96 are potent molecules to induce MHC class I-restricted cytotoxic T cells against antigens present in the cells from which the HSP were isolated. Fusion proteins consisting of mycobacterial Hsp70 covalently linked to antigenic peptide sequences are also capable of generating CTL specific for the peptide-encoded antigens. For effective CTL induction direct binding of the peptide or covalent association of the peptide in the case of antigenic fusion proteins is required. Since mycobacterial Hsp70 and eukaryotic Hsp70 differ significantly in their primary structure, and since gp96 compared to Hsp70 is more efficient in priming antigen specific CTL in our hands, we created fusion proteins consisting of His-tagged eukaryotic gp96 fused C-terminally to various peptide antigens. Here, we used antigenic sequences derived from the established ovalbumin (OVA) and beta-galactosidase (beta-GAL) model systems. We show that in vitro established OVA and beta-GAL specific CTL clones release TNF-alpha and IFN-gamma when incubated with recombinant gp96 irrespective of the antigenic peptide sequences hooked to the C-terminus of gp96. In contrast to gp96 preparations purified from beta-GAL expressing cell lines, recombinant gp96/beta-GAL fusion proteins were not able to generate beta-GAL-specific T cells in vivo. Possible explanations for the lack of antigen-specific immunogenicity of gp96 fusion proteins in vivo are discussed.