We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, 0.12 log₁₀ copies/ml; RT-PCR, 0.05 log₁₀ copies/ml) and after 18 h (bDNA assay, 0.27 log₁₀ copies/ml; RT-PCR, 0.15 log₁₀ copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, 0.002 log₁₀ copies/ml; RT-PCR, 0.02 log₁₀ copies/ml), but it was after 18 h (bDNA assay, 0.09 log₁₀ copies/ml; RT-PCR, 0.09 log₁₀ copies/ml). Only 4% of samples processed after 6 h lost more than 50% (0.3 log₁₀ copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4⁺-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (0.05 to 0.12 log₁₀ copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4⁺-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.