Different features have been associated with low susceptibility to HIV type 1 (HIV-1) infection in exposed seronegative individuals. These include genetic make-up such as homozygosity for the CCR5-Δ32 allele and the presence of HIV-specific CTLs. We studied immune activation and immune responsiveness in relation to HIV-1 susceptibility in 42 high-risk seronegative (HRSN) participants of the Amsterdam Cohort Studies and 54 men from the same cohort who were seronegative at the moment of analysis but later became HIV seropositive. HRSN had higher naive (CD45RO CD27) CD4 and CD8 T cell numbers and lower percentages of activated (HLADR CD38, CD70) CD4 and proliferating (Ki67) CD4 and CD8 T cells, irrespective of previous episodes of sexually transmittable infections. Furthermore, whole blood cultures from HRSN showed lower lymphoproliferative responses than healthy laboratory controls. These data suggest that low levels of immune activation and low T cell responsiveness may contribute to low HIV susceptibility.