The present report provides the first extensive characterization of the OT‐I TCR transgenic line, which produces MHC class I‐restricted, ovalbumin‐specific, CD8⁺ T cells (OT‐I cells). These cells are shown to be positively selected in vivo in H‐2^b C57BL/6 mice and in bm5 mice, which express the K^bm5 mutant molecule. In contrast, OT‐I cells were not selected by mutant K^b molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT‐I cells, as measured by induction of an OVA‐specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.