Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules depends on translocation of cytosolic peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Peptide transport by TAP is thought to include at least two steps: initial binding of peptide to TAP, and its subsequent translocation requiring ATP hydrolysis. These events can be monitored in peptide binding and transport assays. Previous studies have shown that the efficiency of peptide transport by human, mouse and rat transporters varies according to the C-terminals of peptide substrates in an allele and species-specific manner. However, it has not been clear during which step of peptide interaction with TAP selection occurs. We used an assay monitoring the peptide binding step to study the binding affinity of a library of 199 peptides for human TAP and the two major allelic rat TAP complexes. We observed a dominant influence of the C-terminus on peptide binding affinity for all transporters, and highly restrictive selection of peptides with aliphatic and aromatic C-terminals by rat TAP1/TAP2u complexes. The selectivity of peptide binding to rat TAP complexes is in full accordance with published data on selective peptide transport and on control of antigen presentation by rat TAP. These results strongly suggest that (i) peptide selection by TAP occurs exclusively in the initial binding step; (ii) all factors involved in peptide selection by TAP are present in insect cells.