AbstractHuman Leukocyte Antigen (HLA)‐E is a low‐polymorphic non‐classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA‐E polymorphism is related to HLA‐E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA‐E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA‐E*01:01 or HLA‐E*01:03, by using a panel of HLA‐E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide‐induced HLA‐E surface expression levels were higher in PBMC from HLA‐E*01:03 homozygous subjects as compared to PBMC from HLA‐E*01:01 homozygous subjects. HLA‐E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA‐E surface expression seemed to be not only dependent on the HLA‐E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA‐E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide‐induced HLA‐E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA‐E expression is regulated and underlines a new role for extracellular peptides in inducing HLA‐E translation, which may represent a defense mechanism against lytic viral infections and necrosis.