Monoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.