The L‐428 cell line derived from nodular sclerosing Hodgkin's disease was verified to be a human female cell line with surface marker and morphologic characteristics similar to native Hodgkin's cells. Single cells were cloned and subcloned twice to determine the characteristics of the clonogenic L‐428 Hodgkin's cell (resulting in a 10% cloning efficiency). Both mononuclear L‐428 cells and classical Reed‐Sternberg cells arose from solitary cells. The clonogenic cell was the mononuclear Hodgkin's cell, although small abortive colonies sometimes arose from classical binucleate Reed‐Sternberg cells. Cytogenetic and phenotypic analysis supported the clonality of three subclones and indicated, among many findings, consistent abnormalities of the long arm of chromosome 7 (beta‐chain of the T cell receptor) and 14q32 (Ig heavy chain). Distinctive abnormalities of cytogenetics, phenotyping and transforming growth factor‐beta production were exhibited for each clone as well. These observations demonstrate the relationship of the continuum of malignant mononuclear and multinuclear Reed‐Sternberg cells in this cell culture from nodular sclerosing Hodgkin's disease and suggest that a similar relationship exists in native Hodgkin's disease tissue. These observations also support the theory of clonality in Hodgkin's disease and suggest that in vivo contiguous metastasis in the L‐428 Hodgkin's disease patient was most likely accomplished by a Ki‐1 positive small mononuclear cell.