Background. We previously showed that vaccination with one-dose of PR1 and WT1 peptides induces transient anti-leukemia immunity(1). We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. Design and Methods. Eight patients with myeloid malignancies were enrolled in this phase 2 study, and 6 completed 6-injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every 2-weeks. Results. Both high- and low-avidity PR1 or WT1-specific CD8+ T-cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8+ T-cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8+ T-cell frequencies further and in all patients the response was lost before the 6th dose. PR1- or WT1-specific CD8+ T-cells were not detected in bone-marrow samples, excluding their preferential localization to this site. Following a booster injection 3 months after the 6th vaccine dose, no high-avidity PR1 or WT1-specific CD8+ T cells could be detected, whereas low-avidity T cells were readily expanded. Cconclusions. These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8+ T-cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.