Fusion of lysosomes to form a giant cytoplasmic inclusion is a major abnormality expressed by multiple hematopoietic and non-hematopoietic cell types in Chediak-Higashi (C-H) patients. In this study, the extent of involvement of lymphoid cell subpopulations was defined. Purified populations of B cells, natural killer (NK) cells, and helper T cells were obtained from two C-H patients and normal controls by immunofluorescence staining of their blood mononuclear cells with the monoclonal antibodies HB-2, Leu-7, or Leu-3 followed by fluorescence-activated cell sorting. Cytochemical and ultrastructural analyses as well as functional assays were performed to determine whether or not the C-H lysosomal abnormality was expressed in the different lymphocyte subpopulations. B cells expressed the C-H defect following activation and differentiation. All of the Leu-7+ cells and a significant proportion of the Leu-3+ cells displayed the C-H abnormality. These Leu-3+ cells share the NK lineage characteristics of granular lymphocyte morphology and the capacity to bind to NK cell targets. In contrast, the C-H abnormality was not observed in non-NK target-binding cells with T helper phenotype, in which clusters of lysosomes formed a normal Gall body. Moreover, T cell functions were unimpaired in C-H patients. These observations raise the issue of the lineal relationship between granular and nongranular lymphocytes typed as T cells on the basis of cell surface antigen markers.