The lymphoproliferative disease of granular lymphocytes (LDGL) results from chronic proliferation of either CD3- or CD3+ large granular lymphocytes (LGLs). Most CD3+ LGLs have been well characterized as clonal LGL proliferations (LGL leukemia). In contrast, the clonal nature and clinical features of patients with CD3- LDGL have not been defined. In this study, we analyzed seven female patients with CD3- LDGL who were heterozygous at certain X-linked gene loci. Neutrophils and CD3- granular lymphocytes were isolated from peripheral blood. Clonal analysis was performed on genomic DNA from these cell fractions on six patients by conventional Southern techniques using probes to the X-linked genes, PGK and DXS255 (M27 beta). In four patients, three of whom were already studied by Southern analyses of genomic DNA and one in whom there were insufficient amounts of DNA, polymerase chain reaction (PCR)-based clonal analysis was performed with primer pairs flanking the BstXI polymorphism on the PGK gene. In six patients, a polyclonal expansion of CD3- granular lymphocytes was demonstrated and in one the result was indeterminate. In contrast to patients with CD3+ LDGL (LGL leukemia), a clonal disease could not be demonstrated with X-linked markers in patients with CD3- LDGL, suggesting a reactive rather than a neoplastic origin of the lymphocytes in these cases.