This phase II study evaluated the effect of chloroquine on the specific CD8(+) T-cell responses to and the safety of a booster dose of investigational human immunodeficiency virus type 1 (HIV-1) F4/AS01(B) vaccine containing 10 μg of recombinant fusion protein (F4) adjuvanted with the AS01(B) adjuvant system. Healthy adults aged 21 to 41 years, primed 3 years before with two F4/AS01(B) doses containing 10 or 30 μg of F4 (ClinicalTrials.gov registration number NCT00434512), were randomized (1:1) to receive the F4/AS01(B) booster administered alone or 2 days after chloroquine (300 mg). F4-specific CD8(+)/CD4(+) T-cell responses were characterized by intracellular cytokine staining and lymphoproliferation assays and anti-F4 antibodies by enzyme-linked immunosorbent assays (ELISAs). No effect of chloroquine on CD4(+)/CD8(+) T-cell and antibody responses and no vaccine effect on CD8(+) T-cell responses (cytokine secretion or proliferation) were detected following F4/AS01(B) booster administration. In vitro, chloroquine had a direct inhibitory effect on AS01(B) adjuvant properties; AS01-induced cytokine production decreased upon coincubation of cells with chloroquine. In the pooled group of participants primed with F4/AS01(B) containing 10 μg of F4, CD4(+) T-cell and antibody responses induced by primary vaccination persisted for at least 3 years. The F4/AS01(B) booster induced strong F4-specific CD4(+) T-cell responses, which persisted for at least 6 months with similar frequencies and polyfunctional phenotypes as following primary vaccination, and high anti-F4 antibody concentrations, reaching higher levels than those following primary vaccination. The F4/AS01(B) booster had a clinically acceptable safety and reactogenicity profile. An F4/AS01(B) booster dose, administered alone or after chloroquine, induced robust antibody and F4-specific CD4(+) T-cell responses but no significant CD8(+) T-cell responses (cytokine secretion or proliferation) in healthy adults. (This study has been registered at ClinicalTrials.gov under registration number NCT00972725).