HIV-specific T cells response and T cell activation are frequently seen in exposed seronegative individuals (ESN). In this study, we report HIV-specific response and level of T cell activation in ESN partners of HIV-infected patients presenting low or undetectable levels of HIV-RNA. We evaluated 24 HIV-serodiscordant couples. ESN were classified into three categories of exposure to HIV (very low, low, and moderate-high), considering levels of HIV-RNA in their infected partner and frequency of sexual high-risk practices within the last 12 mo. HIV-specific T cell responses and activation levels in T cell subsets were evaluated by flow cytometry. We reported that 54% of ESN had detectable HIV-specific T cells response, being the highest prevalence seen in the low exposure group (64%). Several T cell subsets were significantly increased in ESN when compared with controls: CD4(+)CD38(+) (p = 0.006), CD4(+)HLA-DR(-)CD38(+) (p = 0.02), CD4(+)CD45RA(+)CD27(+)HLA-DR(-)CD38(+) (p = 0.002), CD8(+)CD45RA(+)CD27(+)CD38(-)HLA-DR(+) (p = 0.02), and CD8(+)CD45RA(+)CD27(-)CD38(+)HLA-DR(+) (p = 0.03). Activation of CD8(+) T cells was increased in ESN with detectable HIV T cell responses compared with ESN lacking these responses (p = 0.04). Taken together, these results suggest that persistent but low sexual HIV exposure is able to induce virus-specific T cells response and immune activation in a high proportion of ESN, suggesting that virus exposure may occur even in conditions of maximal viral suppression in the HIV-infected partner.