Abstract The B lymphocytes serving as antibody-forming-cell (AFC) progenitors have been investigated using two different types of separation methods. Sedimentation velocity fractionation was used to separate subsets of B lymphocytes differing primarily in size. Fractionation on a 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-gelatin matrix was used to separate NIP-binding cells, a population highly enriched for cells with surface Ig receptors specific for the NIP hapten. Assessment of the functional capacity of the separated B cells was by culture at limiting dilution in the presence of thymus “filler” cells, using the T-independent antigen NIP-POL (polymerized bacterial flagellin) to induce antibody formation. Splenic AFC-progenitors from both adult conventional and neonatal germ-free mice were a physically heterogeneous population, with activity in small, medium, and large lymphocytes. The cells enriched by NIP-gelatin binding, whether isolated and counted directly or isolated and assayed as AFC-progenitors, were no less heterogeneous. These NIP-binding cells resembled in sedimentation characteristics the overall B-cell and overall NIP-specific AFC-progenitor populations, except for some relative enrichment of medium-sized cells ( S value, 5.5 mm/hr). The small ( S value, 3.4 mm/hr) B-cell region of adult mouse spleen contained both NIP-binding cells and cells responsive as AFC-progenitors in the microculture assay. This contrasts with the results of the in vivo adoptive immune assay, where the smaller B-cell region is unresponsive in unprimed adult animals.