In multiparameter fluorescence-activated cell sorter studies presented here, we use the highly fluorescent protein phycoerythrin (PE) as an immunizing antigen and as a fluorescent probe to reveal and isolate antigen-binding memory B cells. We demonstrate directly that memory B cells in spleens from PE-primed mice stain brightly with PE and produce strong IgG1 anti-PE responses when sorted and cultured with L3T4+ helper T cells. That is, as few as 500 sorted PE-binding cells per culture well are sufficient to produce high-level anti-PE responses, presenting antigen in a form that induces helper T cells. Three-color immunofluorescence studies reveal that cells staining brightly with PE reside primarily in a population of B220+(IgM,IgD)- cells and comprise 0.02-0.05% of the spleen cells. Complementary studies with 2,4-dinitrophenyl memory B cells show that whereas this B220+-(IgM,IgD)- population (which comprises only 1-2% of the spleen cells) gives high-affinity anti-dinitrophenyl responses, the predominant B220+(IgM,IgD)+ population yields only low-affinity antibody. Finally, we find that although both populations have similar Ia levels, cells in the high-affinity fraction have greatly reduced levels of surface immunoglobulin.