Susceptibility of PHA/rIL-2-stimulated PBMC from 14 healthy blood donors for NSI HIV-1 infection was analyzed in relation to CCR5 expression and β-chemokine production. After 1 week of culture in the presence of rIL-2, but not at the moment of inoculation, CCR5 surface expression was positively and β-chemokine production was inversely associated with susceptibility to NSI HIV-1 infection. Surprisingly, no association was observed between CCR5 genotype and in vitro NSI HIV-1 susceptibility, which was in agreement with similar levels of CCR5 surface expression and β-chemokine production in CCR5Δ32/+ and CCR5 +/+ PBMC after PHA/rIL-2 stimulation. In contrast to what was observed in vitro, CCR5 genotype did associate with CCR5 surface expression levels in vivo in resting as well as in activated CD4+ T cell populations that were identified by the expression of CD45RO, CD27, HLA-DR, and CD69. The association between CCR5 expression and susceptibility to infection by NSI HIV-1 observed in vitro might offer an explanation for the in vivo observed protective effect of CCR5 polymorphisms that influence CCR5 expression on disease progression.