Positive selection is a process that ensures that peripheral T cells express TCR that are restricted to self-MHC molecules. This process requires both self-MHC and self-peptides. We have recently established a TCR transgenic mouse model (C10.4 TCR(trans)+) in which the transgenic TCR was selected on the nonclassical MHC class Ib molecule H2-M3 in conjunction with a physiologically occurring peptide derived from the mitochondrial NADH- dehydrogenase subunit 1 gene (9-mer peptide). Here, the specificity of positive selection of C10.4 TCR(trans)+ T cells was examined using a fetal thymic organ culture system. We demonstrated that at low peptide concentrations, shortening the NADH-dehydrogenase subunit 1 gene 9-mer peptide or mutating its surface-exposed side chains severely impaired its ability to induce positive selection. We concluded that under physiological conditions positive selection of C10.4 TCR(trans)+ T cells was highly specific and occurred at low epitope densities.