Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10 2 and 10 7 RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R 2 ؍ 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.The viral RNA level in the plasma or serum of human immunodeficiency virus type 1 (HIV-1) infected individuals has become the most important marker for several aspects of an HIV-1 infection. The HIV-1 RNA level is predictive for disease progression in untreated infected individuals (7,13,17,18,25), and it is the best marker for monitoring therapy effects (14,20,31). In addition, the presence of viral RNA in the serum of newborn children is used as evidence for mother-tochild transmission, since maternal antibodies present in infant serum hamper antibody-screening assays. In this decade the number of newborn infants screened will increase as soon as cheaper and easier assays become available for use in developing countries, where they are needed most. The presence of viral RNA will also be one of the most important markers for monitoring breakthroughs in vaccine studies due to the presence of viral antibodies in sera as a result of vaccination. In addition, vaccines that do not have a strict preventive eff...