The successful application of an improved enzyme-linked immunosorbent assay (ELISA) to the specific detection and quantitation of feline leukemia virus (FeLV) is presented. The methods described can detect FeLV antigens at the picogram/milliliter level using a fluorometric assay for peroxidase-linked antibody. The use of a fluorogenic substrate resulted in at least a 9-fold increase in sensitivity of the peroxidase assay. FeLV antigens could be specifically detected in chronically infected cells as well as in feline sarcoma virus-transformed nonproducer mink cells which only partially express viral proteins.