We have adapted our established Alu PCR assay for proviral DNA and PCR for total cellular DNA to a real-time PCR format and applied these to human immunodeficiency virus (HIV)-positive specimens collected for routine determination of the plasma viral load (pVL). In a cohort of five patients, measurements of integrated viral load (iVL) and cell-associated viral load (cVL) in CD4(+) cells isolated by a single positive selection step were not indicative of HIV DNA levels in the circulation, and further analysis was performed on peripheral blood mononuclear cells (PBMC). In a cohort of 46 samples total cVL was quantitated in most samples, but iVL could be quantitated in only 47.8%, since in 26% iVL was undetectable and in 21.7% the results were invalid due to high levels of unintegrated HIV DNA. There was no correlation of cVL or iVL with pVL, CD4 count, or duration of successful antiretroviral treatment. Out of 26 patients with undetectable pVL, 4 patients failed therapy within the subsequent 12 months and had higher than average iVL, but this was not the case for cVL. Among nine patients with long-term undetectable pVL, no consistent decline in cVL or iVL was seen with time, and changes in cVL and iVL within a patient could be concordant or discordant. These results show that cVL and iVL can be coordinately measured in PBMC from clinical samples but do not correlate with pVL, CD4 counts, or length of suppressive antiretroviral therapy. Interestingly, a high iVL (but not a high cVL) in patients with undetectable pVL was associated with subsequent treatment failure.