The lung carcinoma cell line Calu3, which overexpresses the c-erbB-2 oncogene, was stably transfected with antisense (AS) cDNA constructs encompassing different regions of the c-erbB-2 gene. Transfected cells were analyzed for their tumorigenic properties in vitro and in nude mice. Two independent clones, AS F1 (low erbB-2 expressor) and AS B12 (high erbB-2 expressor), as well as the polyclonal Calu3/AS 5', were selected for these analyses. In Calu3/AS 5' transfected cells and in the AS F1 clone, c-erbB-2 RNA and protein levels were lower than those detected in the parental cell line and the AS B12 clone. Anchorage-independent growth and tumor take were also significantly reduced. Furthermore, cells derived from primary tumors of Calu3/AS 5', AS F1 and AS B12 lost the AS c-erbB-2 DNA insert but retained the gene for G418 resistance. Our results suggest that a correlation between c-erbB-2 overexpression and tumorigenicity may exist in the Calu3 lung carcinoma cell line.