Feline immunodeficiency virus (FIV) affects between 1 and 14% of the cats in the United States. Although infected cats may be asymptomatic for many years, an AIDS condition ultimately develops, leading to opportunistic infections, cancer, and wasting. No treatment exists for FIV, other than for the secondary diseases that develop. Until recently, the only method for avoiding FIV infection was to prevent cats from having exposure to FIV-infected cats. Isolation of infected cats was accomplished by testing cats for antibodies against FIV, a highly reliable indicator of infection. The recent release of Fel-O-Vax FIV®, the world’s first vaccine against FIV, was a scientific breakthrough in terms of overcoming the challenges of FIV vaccination. However, the vaccine also opened a Pandora’s box regarding diagnosis of FIV, because the vaccine interferes with all of the currently licensed FIV diagnostic tests based on detection of antibodies. Vaccinated cats produce antibodies that are indistinguishable from those used for diagnosis of FIV infection. Polymerase chain reaction (PCR) has been suggested as an alternative testing methodology, but virtually nothing is known about the sensitivity, specificity and overall diagnostic performance of the currently available tests. Various PCR tests have been shown to be incapable of detecting some FIV strains, due to the marked variability of the viral genome. The sensitivity of PCR for low amounts of virus may lead to false positive results if even minor contamination occurs during handling of samples. Failure to identify infected cats (false negative) may lead to inadvertent exposure and transmission of FIV to uninfected cats. Misdiagnosis of FIV in uninfected cats (false positive) may lead to the inappropriate euthanasia of cats or of kittens born to vaccinated queens. This is an especially problematic issue in shelter medicine, since confirmatory testing is frequently impractical due to financial and logistical limits. In this dawning era of FIV vaccination, it is essential to evaluate all of the currently available FIV diagnostic tests to determine which are the most reliable and where the pitfalls lie. This study will evaluate test results for all of the currently available FIV diagnostic tests in FIV-free, FIV-vaccinated, and FIV-infected cats against a gold standard of viral isolation.