Four human cell lines were established from biopsy specimens of squamous cell carcinomas of the larynx (TR131 and TR138), tongue (TR126), and buccal mucosa that had infiltrated a lymph node (TR146). All 4 lines readily formed colonies on a plastic substratum, but they were virtually incapable of forming colonies in an anchorage-independent semisolid support system of soft agar (cloning efficiencies, less than 0.02%). The proliferation of this group of tumor-derived cell lines, therefore, appeared to be highly anchorage dependent. Keratin filaments could be visualized in each line by indirect immunofluorescence with the use of polyclonal or monoclonal antibodies to keratins; staining with monospecific antibodies indicated that 3 of the 4 lines expressed simple epithelial keratins 8 and 18, whereas 1 of the 4 also expressed keratin 19. A panel of lectins revealed characteristic localization patterns distinct from those observed on other epithelial cell lines. Cells from 3 lines (TR131, TR138, and TR146) inoculated into nude mice (nu/nu) produced cystic nodules or unequivocal tumors having a histology indicating a squamous cell origin for the injected cells. Electron microscopy demonstrated that the cell lines covered a spectrum of differentiation capability ranging from the undifferentiated monolayer cultures of TR126 to the rather well differentiated, stratified cultures of TR131.