Positive selection of functional CD8+ T cells expressing an MHC class I-restricted T cell receptor can be induced in fetal thymus organ culture by class I-binding peptides related to the antigenic peptide ligand. Peptides that act as antagonist or weak agonist/antagonist ligands for mature T cells work efficiently in this regard. In the present study, we have investigated whether low concentrations of the original agonist peptide, or variants that still have a strong agonist activity can also mediate positive selection. The antigenic peptide did not induce positive selection at any concentration tested. A strong agonist variant was capable of stimulating the differentiation of TCRhi CD8+ cells, giving the appearance of phenotypic positive selection. However, these cells lacked biological function, since they could not proliferate in response to antigen. The most efficient positive selection resulted with ligands that did not activate mature T cells or stimulate negative selection.