In our effort to delineate factors that govern the ability of non-small cell lung carcinoma (NSCLC) to form monolayer cell lines, we have attempted to derive monolayer cell lines from the primary cultures of 29 unselected human NSCLCs. Eight new lines were obtained. Cell lines were easier to establish from poorly differentiated tumors, especially adenocarcinomas. One cell line was from a large cell neuroendocrine carcinoma. All cell lines were aneuploid, and they exhibited heterogeneous nutritional requirements for growth in vitro. Cell line-forming primary tumors demonstrated higher mean messenger RNA expression levels for transforming growth factor-alpha and c-met proto-oncogene than did tumors that failed to form cell lines. Although a high level of c-myc expression was correlated with the ability of NSCLC cell lines to form xenograft tumors in nude mice, it was not correlated with the ability of primary tumors to establish cell lines. The results suggest that autocrine growth loops play important roles in the ability of NSCLC cells to proliferate continuously in monolayer culture. The fact that the overexpression of transforming growth factor-alpha in NSCLCs has been negatively correlated with patient survival and that most cell lines can be established only from poorly differentiated carcinomas may provide the explanation for a previous report that the capability for cell line establishment constitutes a negative prognostic indicator for patient survival. However, when the genotype and phenotype of the cell lines were compared with those of their corresponding primary or xenograft tumors, the tumor cells that grew continuously as a cell line often represented a selective subpopulation of the heterogeneous neoplastic cells in the primary tumors. This finding should be taken into consideration when cell lines are used to evaluate the chemo- and radiosensitivity of tumor cells.