Three clonal sub-populations, GRU-IA, GRU-IB, and GRU-IC, isolated from the human epithelioid sarcoma cell line GRU-I, were characterized morphologically, cytogenetically and with regard to proliferation kinetics. Immunocytochemically, major differences became evident in the expression of cytokeratin 18 and neurofilament proteins, which are indicative for epithelial and neural differentiation respectively. Vimentin, a mesenchymal differentiation marker, however, could be detected in all tumor cells of each sub-population. Laminin, a major compound of basement membranes, formed abundant intercellular network-like patterns in GRU-IB and GRU-IC, whereas GRU-IA was characterized by a diffuse intracellular reaction, suggesting a disorder in laminin secretion. Cytogenetically, all sub-populations proved to be DNA-aneuploid, the DNA index ranging from 1.4 to 1.5. Proliferation analysis revealed doubling times ranging from 13 (GRU-IC) to 19 hr (GRU-IA). These strictly defined clonal sub-populations provide a valuable tool for further investigations of the biological behavior of human epithelioid sarcoma with special regard to tumor heterogeneity.