An improved quantitative polymerase chain reaction (qPCR) method based on a combination of real-time detection and the 5"-3" nuclease activity of the Taq DNA polymerase was developed to quantify the provirus load of feline immunodeficiency virus (FIV), a lentivirus of veterinary importance and an animal model for AIDS research. Two fluorogenic probes were designed to detect FIV provirus in genomic DNA of peripheral lymphocytes and tissues infected with different FIV subtypes. The most sensitive assay can detect one copy of FIV provirus. The assay showed excellent precision within-run and between-runs. Comparison of the TaqMan system with a conventional seminested PCR assay revealed a comparable detection limit and good correlation. Furthermore the design of the two probes allowed the detection of various FIV isolates of clade A and B.