Abstract Enumeration of lymphocyte subpopulations by combined use of flow cytometry and monoclonal antibodies is influenced by the separation method used. T or B lymphocyte antigen frequencies do not differ in samples of whole blood or after separation on Ficoll-Paque or Percoll, but there is a significant increase of Leu7 + Leu11 + lymphocytes showing strong natural killer activity at the sample-separation medium interface. At the bottom of the tubes a selective loss of OKT8 +, Leu11 − cells is found; at this level cytotoxicity is very low. Our data suggest that Leu7 + cells could be subdivided into 2 subpopulations differing in reactivity with the monoclonal antibody to Leu11, natural killer activity and density. Differences observed in the percentages of Leu7 +, Leu11 + lymphocytes between whole blood and separated cells could be due to an enrichment produced by centrifugation or to the presence in whole blood of a factor interfering with antibody binding.