Measurement of HIV-1 viral load in plasma is an important marker of disease progression and efficacy of antiretroviral therapy. Real-time polymerase chain reaction (PCR) offers an opportunity to develop more affordable alternative viral load assays. This article reports on the development of a novel real-time reverse-transcriptase (RT)-PCR assay for quantitation of HIV-1 RNA copies. This assay utilizes the LightCycler (version 2) real-time PCR platform and light upon extension (LUX) primer for specific detection of amplicons. An external standard (ES) for quantitation of viral RNA represents an in vitro transcribed RNA. The LUX assay shows a wide linear (R2 = 0.99) dynamic range from 4 x 10(6) to 4 x 10(2) copies/mL. Analytical sensitivity of the assay is 4 x 10(2) copies/mL of ES RNA. Intra- and inter-assay variability of the LUX assay was less than 0.5log(10) copies of ES RNA (i.e., no clinically significant variability was found). Virology quality assurance (VQA) HIV-1 RNA copy controls were used to validate ES and preliminarily evaluate the assay performance. This feasibility study demonstrated that the LUX assay is sensitive, reproducible, and compares well to the Roche Amplicor tests used for characterization of the RNA copy controls. These results suggest further evaluation of the LUX assay using a large cohort of well-characterized samples from HIV-1 positive individuals.