The question of when immune reconstitution of cytomegalovirus (CMV)-specific CD8 T cells occurs after hematopoietic cell transplantation and, more specifically, to which CMV targets this immunity is likely to be directed remains poorly understood. The dependence of immune reconstitution on CMV reactivation is even less clear. To better understand these events, 44 CMV-seropositive HLA-A*0201 subjects were followed up at approximately days 40, 90, 120, 150, 180, and 360 after hematopoietic cell transplantation for CMV immunity as measured by 2 types of assays (1) an HLA-A*0201 tetramer-binding assay for both CMV pp65 (pp65) and immediate-early 1 (IE-1) or (2) intracellular cytokine interferon gamma responses induced by pp65 or IE-1-derived peptides. To verify the reliability of IE-1-specific assays relative to the pp65-based assays, a pilot study first compared the development of IE-1-specific immunity in a subgroup by using multiple HLA-A*0201-restricted peptides, and then these recipients were followed up for 1 year for immunologic function and for CMV infection. The IE-1-specific response occurred to each of the 3 HLA-A*0201-restricted peptides studied (IE-1-256, -297, and -316), and there was no predominant IE peptide response. However, the immunodominant HLA-A*0201-restricted pp65 peptide was recognized significantly more frequently than these IE-1 peptides. When this was compared with the occurrence of CMV infection, the overall immune reactivity, as measured by the mean or median number of CD8+ T cells reactive to either pp65 or IE-1 peptides by intracellular cytokine or tetramer binding assay, was not significantly different in those with and without CMV infection. For patients who demonstrated reconstituted immunity to CMV at 1 year, all were reconstituted by 6 months, and the timing of the first observed immune reactivity to either of the pp65 or the IE peptides was not different in those with and without detectable CMV infection.