Optimal conditions of culture and assay for identification of feline immunoglobulin-secreting mononuclear cells were determined for the staphylococcal protein A-reverse hemolytic plaque assay (SpA-RHPA). Hemolytic plaques were most distinct and numerous when peripheral blood mononuclear cells were stimulated with 6.9 micrograms/ml pokeweed mitogen for 7 days. Immunoglobulin-secreting cells were identified morphologically within a zone of hemolysis utilizing a 1:5 dilution of rabbit anti-cat IgG and a 1:30 dilution of guinea pig complement as developing reagents. The SpA-RHPA system should contribute to an understanding of normal feline T- and B-lymphocyte interactions and will likely aid in the identification and understanding of immune cell dysfunctions associated with chronic feline leukemia virus infection.