Dendritic cell (DC) vaccination, i.e. the adoptive transfer of antigen-loaded DC, is still at an early stage and requires standardization. In this study, we investigated the exogenous loading of monocyte-derived DCs with HLA class I- and II-restricted peptides, as despite widespread use, little effort has been put into its pre-clinical validation. We found that only mature DCs (m-DC) but not immature DCs (im-DC) could be sufficiently loaded with exogenous class I-restricted peptides and were by far superior in expanding CD8(+) primary (Melan-A.A2 peptide-specific) and recall [Influenza matrix peptide (IMP) A2-specific] T cell responses. Primary stimulation with peptide-loaded im-DCs even down-regulated antigen-specific T cell responses. Our results indicate that stimulation with m-DCs is superior in terms of quantity and quality compared with im-DCs, supporting their preferred use in clinical DC trials. Loading of m-DCs with high (10 microM) concentrations generated clearly more Melan-A effectors than loading with 1 or 0.1 microM without any negative effect on the quality (affinity) of the resulting T cells. In contrast to the findings with the Melan-A peptide loading with 10 microM IMP was counter-productive, induced apoptosis and yielded fewer specific T cells of inferior affinity as compared with loading with 1 or 0.1 microM. In sharp contrast to the situation for HLA class I, much higher levels and longer half-lives of peptide-HLA class II complexes were obtainable upon loading of im-DCs with exogenous peptide, but m-DCs were functionally preferable to induce T(h)1 responses in vitro. Another surprising finding was that, while presentation to T cells upon simultaneous loading of several peptides with highly varying affinities and competing for the same class I or II molecule was possible, in priming experiments peptide competition clearly inhibited T cell induction. Although peptides will obviously vary in their individual properties, our study clearly points to some important principles that should be taken into account.