Feline immunodeficiency virus (FIV) affects from 1% to 30% of cats worldwide. The infection is incurable and results in severe immunosuppression, cancer, and wasting syndromes. The diagnosis of FIV infection in cats has relied on detection of virus-specific antibodies in blood. Until recently, the only method for prevention of FIV infection was identification of infected cats followed by euthanasia or separation from uninfected cats. In July 2002, the first vaccine became available for prevention of FIV. Vaccinated cats produce antibodies that are indistinguishable from those produced by naturally infected cats, thereby invalidating currently used antibody-based diagnostic assays. The polymerase chain reaction (PCR) assay has been proposed as an alternative method for determining whether FIV antibody-positive cats are infected or vaccinated. However, we recently completed a study that revealed the diagnostic performance of FIV PCR tests offered by commercial laboratories is poor. Virus culture is accurate, but is too costly and technically demanding to be offered as a routine test. Consequently, there is no reliable diagnostic assay currently available for verification of the true status of FIV antibody-positive cats. Recently, we have developed a PCR assay that accurately identified uninfected and infected cats in preliminary studies. The objectives of this proposal are: 1) to develop a cost-effective and timely virus culture assay for routine use, and 2) to determine the overall diagnostic performance of the new PCR assay on blood samples from uninfected and FIV-infected cats.