OBJECTIVE—Islet-reactive CD8+ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS—We took advantage of a recently validated islet-specific CD8+ T-cell γ-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2+ adult type 1 diabetic patients close to diagnosis and at a second time point 7–16 months later. RESULTS—CD8+ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60–67 to 20% (P < 0.02). The previously subdominant IA-2206–214 and IGRP265–273 peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS—Shifts both in frequency and in immunodominance of CD8+ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.