Two new permanent cell lines derived from squamous cell carcinomas of the tongue, CAL 27 and CAL 33, have been established in culture. Both cell lines were isolated in standard culture media without epidermal growth factor or fibroblast feeder layer to avoid obtaining clones of more differentiated cells. Analysis of the morphology, ultrastructure, karyotype and immunohistochemical properties of these two cell lines demonstrated that they are both well characterized, uncontaminated by HeLa cells, and do in fact correspond to transformed epithelial cells that have conserved certain characteristics of the original Malpighian epithelium. CAL 27 and CAL 33 have relatively long doubling times (35 and 43 h respectively). Their response to 14 drugs used for cancer chemotherapy was evaluated by a short term assay based on tritiated thymidine incorporation after exposure to the drugs. CAL 27 was more resistant than CAL 33 in all cases but one. Although cytogenetic examination revealed both lines to be malignant, neither CAL 27 nor CAL 33 produced colonies in soft agar; both lines were tumorigenic after inoculation into nude mice. This study clearly demonstrates the diversity of cancers of a given histologic form, in agreement with the diversity noted previously in vivo. Isolated without the use of any selection criteria, these cell lines constitute appropriate models for the study of human tumors.