Three new techniques have been introduced for the phenotypic analysis of human thymocytes and T cells. Monoclonal antibodies of different subclasses (i.e. IgG1 and IgG2) were used in various combinations and labelled with subclass-specific second layers conjugated to different fluorochromes. Tissue sections were studied with combinations of monoclonal and conventional reagents, and anti-mouse Ig-coated macrobead columns were used to separate antibody-tagged cells. With the help of these simple techniques, four stage of thymocyte differentiation (prothymocyte, cortical, intermediate and medullary cell types) can be described in detail and the microanatomical relation of T cell subsets with other cell types studied.