Background: Human T-lymphotropic virus type I (HTLV-I) is linked etiologically with adult T cell leukemia/lymphoma, HTLV-I-associated myelopathy/tropical spastic paraparesis and other inflammatory diseases. HTLV-1 infection has been widely documented in the world, existing endemic areas in our country with virus circulation demonstrated. HTLV virus integrates into the host cell genome as a provirus. In contrast to HIV, cell-free viremia in plasma is not a prominent aspect of HTLV-associated diseases. Due to this characteristic, the most appropriate method for molecular diagnosis is the detection of proviral DNA by PCR. Moreover, real-time PCR is the only method able to quantify the proviral load of virus present in the sample. However, nowadays there are not commercially real-time PCR techniques available to quantify this virus. Proviral load may be important in the progression of HTLV-associated disease. Objectives: To develop and implement a real-time PCR technique for detection and quantification of HTLV-1 proviral DNA in peripheral blood mononuclear cells (PBMCs) and to use it to quantify the provirus in blood samples from asymptomatic and symptomatic individuals infected with HTLV-1. Materials and Methods: A real time quantitative PCR assay using SYBR Green intercalation dye was established. Primers targeting the tax region were standardized against MT2 cell line DNA for HTLV-1. HTLV-1 copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. We assessed the limit of detection and quantification and the intra- and inter-assay reproducibility of the assay. Developed methodology was used to measured proviral load in PBMCs in a cohort of 50 HTLV-1 seropositive individuals. Results: The assay has a lower limit of detection of a single copy, reaching 100% of sensitivity when 5 copies of provirus were tested per reaction. Proviral load for HTLV-1 infected patients ranged from 2.2×102 to more than 8.3×104 copies/106 PBMCs. Conclusion: The assay has excellent dynamic range from 105 to 100 copies/reaction and good intra- and inter-assay reproducibility. The sensitivity and high dynamic range allow determination of a broad range of HTLV-1 proviral load in clinical subjects. This assay is a valuable tool that will facilitate the study of the relationship between proviral load and pathogenesis.