A reverse hemolytic plaque-forming cell (PFC) assay for the enumeration of immunoglobulin-(Ig)secreting cells in nonimmunized rabbits was developed by using erythrocytes coated with anti-Ig antibody. These indicator cells lysed after reacting with the secreted Ig antigen and complement. The anti-Ig antibodies used were specific for the b4 or b5 allotypic specificities, which are antigenic determinants on the kappa chain of rabbit Ig and controlled by alleles at the b locus. We coated erythrocytes with anti-b4 or anti-b5 antibody, using hybrid antibody with dual specificity to sheep red blood cells and to the b4 or b5 kappa chain. The coated erythrocytes were plated with cells from various lymphoid organs of nonimmunized rabbits. These lymphoid cells formed allotype-specific plaques, i.e., the cells of b4 homozygous rabbits formed hemolytic plaques with anti-b4 but not with anti-b5-coated erythrocytes and vice versa. In eight nonimmunized rabbits, 0.06-0.3% of the spleen cells (590-2900 PFC per 10(6) cells) secreted the b locus Ig allotypes. In three nonimmunized rabbits, 320-590 PFC per 10(6) cells were counted in the lymph node or bone marrow; 26-40 PFC per 10(6) cells were detected in the thymus. Since the b4 and b5 specificities are present on approximately 90% of the serum Ig, our results indicate that less than 1% of the lymphoid cells in nonimmunized rabbits secrete Ig at any one time. By this reverse PFC assay, Ig-secreting cells were for the first time detected as antigen-secreting cells. This assay can be applied to the detection of cells secreting other antigens.