MHC class I molecules bind short peptides for presentation to CD8⁺ T cells. The determination of the three‐dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class I a molecules expressed in the TAP2‐deficient mouse cell line RMA‐S: RT1‐A1^c, which carries unusual key residues at both ends of the groove, and RT1.A^a which carries the canonical residues. Temperature‐dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7 – 15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2‐K^b, 8 – 13‐mer and H2‐D^b, 9 – 15‐mer peptides). The rat MHC class Ia molecule, RT1‐A^a, revealed a preference for 9 – 15‐mer peptides, whereas RT1‐A1^c showed a more stringent preference for 9 – 12‐mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.