We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 × 10⁵cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for ∼10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells.