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Modification of Sepharose 6MB affinity beads by conjugation with CIBA blue dextran and albumination together with siliconization and modification of the bed supports of glass chromatography columns permitted the construction of affinity columns with low non-specific binding characteristics. When used with staphylococcal protein A as the affinity ligand to fractionate antibody-coated lymphocytes, non-specific adherence was reduced to 2--3% of the viable input cells and essentially the entire input cell number could be recovered in viable, functional form. The capacity of the columns permitted quantitative separation of T and B lymphocytes in 250 microliter amounts at cell densities up to 5 X 10(8)/ml on 1.2 ml columns. Once used, the columns may be regenerated and reused numerous times without alteration of their characteristics. The method thus appears useful for preparative separation without loss of large numbers of highly purified cells having normal in vitro reactivity.