Circulating Recombinant Form (CRF) AE is the most common HIV-1 subtype found in Thailand which causes AIDS epidemic in Thailand. The virus sequence of AE subtype is different from subtype B which commonly found in developed countries. Cytotoxic T Lymphocytes (CTLs) are considered as an important protective immunity against HIV infection. Vpr arrests cell cycle at the G2/M phase that can increase virus production. Vpu facilitates the budding of new virus particles from infected cells and also enhances the degradation of CD4. There are no CTL studies of HIV-1 CRF01_AE-specific CTL epitope mapping directed against both Vpu and Vpr. So this study may provide additional useful data for the research and development HIV/AIDS vaccine in Thailand. This study is to demonstrate Vpu and Vpr-specific CTL responses and to map the corresponding epitopes in 20 untreated HIV-1 infected Thais by using IFN-gamma ELISPOT assay. HIV-1 CRF01_AE based Vpu and Vpr peptides 15-20 amino acids in length, and overlapped by 10 amino acids were used for this study. There are 4 pools of Vpr and Vpu peptides, each contains 5 truncated peptides except the last pool contains 3 Vpu peptides. The positive cut-off of ELISPOT was defined as the IFN-gamma spot-forming unit (SFU)/10[superscript 6]PBMCs after the background subtracted is [is more than or equal to] 100 SFU/10[superscript 6]PBMCs and > 2.5 folds higher than negative control. Patients who showed positive results were then further identified for Vpr and Vpu specific epitope (s) recognition. Eighteen out of 20 showed CRF01_AE infection by subtyping. Nine out of 18 (50%) AE-infected patients showed IFN-gamma ELISPOT positive responses to Vpr pooled peptides. The magnitude of responses ranged from 132-1,800 SFU/10[superscript 6]PBMCs (median 470 SFU/10[superscript 6]PBMCs). Five out of 18 AE-infected patients (28%) showed positive responses to Vpu pooled peptides. The magnitude of responses ranged from 124-908 SFU/10[superscript 6]PBMCs (median 394 SFU/10[superscript 6]PBMCs). 7/18 HIV-1 CRF01_AE patients (39%) who showed positive responses to Vpr pooled peptides showed positive results to Vpr individual peptides. The magnitude of responses ranged from 114-1,572 SFU/10[superscript 6]PBMCs (median 880 SFU/10[superscript 6]PBMCs). 3/17 HIV-1 CRF01_AE patients (18%) showed positive results to Vpu individual peptides with the magnitude of responses ranged from 136-680 SFU/10[superscript 6]PBMCs (median 306 SFU/10[superscript 6]PBMCs). However, in this study there are 2 possible novel epitopes : one in Vpr and the other in Vpu were found. Further characterization of these epitope is warranted.