Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-alpha were produced (E6T cells). To define the role that TGF-alpha production played in induction of tumorigenicity, a non-tumorigenic TGF-alpha-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-alpha (E2alpha cells). Transfected clones produced TGF-alpha at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-alpha antisense vector (E6TA cells) expressed only 6% of the TGF-alpha level of the parental cells. Clones of E6T, E6TA, E2 and E2alpha were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2alpha cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2alpha and E6TA cells, and ErbB-2 were greatly overexpressed in an E2alpha clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2alpha TGF-alpha-producing cells, indicating that the lower TGF-alpha levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-alpha sufficient to activate downstream signalling by ErbB-2 containing active heterodimers.