Feline immunodeficiency virus (FIV) is an AIDS-inducing lentivirus that infects domestic cats worldwide. Because of its clinicopathologic similarities to human immunodeficiency virus type 1 (HIV-1) infection, the FIV/cat infection system is a valuable animal model for investigating comparative aspects of HIV-1 biology. An assay that detects quickly and efficiently FIV RNA in relatively small volume samples of feline blood or other body fluids would be of benefit in studies of viral transmission and antiviral interventions. Nucleic acid sequence based amplification (NASBA) technology is particularly suited for the detection of RNA in a variety of body fluids. In this report, the development of two rapid, sensitive and versatile NASBA formats is described for the detection of FIV gag RNA in plasma from infected cats. RNA detection by either format was unaffected by the presence of feline plasma. The limits of detection were at least 200 copies of input RNA for both formats. Results from seropositive and seronegative feline plasma samples were clearly distinguishable. These results demonstrate that NASBA provides a rapid and sensitive alternative to RT-PCR and culture isolation for detecting FIV RNA in infected feline plasma.