Peritoneal exudates from BALB/c mice rejecting C57BL ascites lymphoma EL4 are a rich source of cytolytic effector lymphocytes (CL); however, these preparations are still contaminated even after removal of adherent cells with other mononuclear cells which do not appear to be cytolytic. The relationship of the cytolytic and "non-cytolytic" cells to graft rejection in vivo is not completely understood. We have used anti-fluorescein (α-FL) columns to separate sensitized lymphoid populations into fractions enriched or depleted in cytolytic activity. EL4 were directly labeled with fluorescein isothiocyanate (FL-EL4), centrifuged with CL and the mixture was applied to a column of horse α-FL antibody conjugated to Sepharose 4B. FL-EL4 and lymphocytes bound to them were retained on the column, while non-bound lymphocytes were collected in a medium wash (passed cells). CL bound to FL-EL4 were then eluted with EDTA (eluted cells). Cytolytic activity of the two fractions was compared to that of the unfractionated population in an in vitro 51Cr release assay. Passed cells were consistently depleted in cytolytic activity compared to unfractionated cells or manipulation controls reaching 100% depletion in some experiments. Enrichment of cytolytic activity in eluted populations was frequently but not invariably observed. Rate of cytolysis was used as a measure of cytolytic activity in fractionated populations. Specificity of binding was investigated in reciprocal experiments using CS7BL/6J effectors raised against BALB/c lymphoma RL♂ 1. Viability of recovered cells was high and the procedure was rapid, efficient and versatile. In contrast to monolayer cellular immunoabsorbents, contamination of fractions with absorbing cells was consistently less than 5%. Both enriched and depleted populations are available for further study of surface markers and function.