We compared the sensitivity and specificity of versions 1.0 and 1.5 and a modified version 1.5 of the AMPLICOR HIV-1 MONITOR ultrasensitive RNA assay (Roche, Indianapolis, IN) by using a virus stock dilution series and plasma samples from HIV-1-infected and uninfected subjects. The modified assay was linear and consistently positive down to 12 copies per milliliter vs 25 copies per milliliter for the other 2 assays. Versions 1.0, 1.5, and modified 1.5, respectively, detected 9 (23%) of 39, 11 (28%) of 40, and 43 (61%) of 71 replicates of a 4-copy-number and 11 (28%) of 40, 17 (46%) of 37, and 88 (90%) of 98 replicates of a 10-copy-number standard. Of 44 patient samples with undetectable levels using version 1.0, 32 (73%) had detectable levels on the modified assay, and 5 (25%) of 20 had detectable levels on version 1.5. None of the assays detected HIV-1 RNA in HIV-1 antibody-negative samples. The modified version 1.5 of the RNA assay is more sensitive for detecting HIV-1 RNA in significantly more patients than are versions 1.0 and 1.5.