A quantitative and sensitive measure of human immunodeficiency virus type 1 (HIV-1) replication is quantitative real-time polymerase chain reaction (PCR). Real-time PCR using SYBR green I and oligonucleotide primers that amplify early, intermediate, and late products of reverse transcription were optimized to measure HIV-1 replication of clade A, B, C, and D HIV-1 isolates in peripheral blood lymphocytes and in both transformed and viral-transformed CD4+ lymphocyte cell lines. Real-time PCR can detect HIV-1 replication as early as 1 hr postinfection and demonstrates that in established cell lines cDNA can be detected as early as 4 hr postinfection. The first round of HIV-1 replication in established cell lines is complete between 12 and 24 hr postinfection. Furthermore, real-time PCR can detect HIV-1 replication in fewer than 0.1% of cells. Patient isolates replicated at different rates in peripheral blood lymphocytes, with viral cDNA peaking between 48 and 120 hr, depending on the virus being studied. Real-time PCR differentiated the mechanisms of action of drugs targeted at HIV-1 entry, reverse transcription, and proteolytic processing and identified differences in the kinetics of reverse transcription between zidovudine-sensitive and zidovudine-resistant HIV in the presence of zidovudine. In summary, real-time PCR using SYBR green I dye is a sensitive, quantitative, and reproducible measure of replication kinetics for a variety of group M HIV-1 isolates.