We conducted an experiment to determine whether expression of transforming growth factor-alpha (TGF-alpha) enhances tumorigenicity in a low-tumorigenicity rat bladder carcinoma cell line and whether it is sufficient to induce a tumorigenic phenotype in a nontumorigenic rat bladder cell line. D44c cells (which are nontumorigenic) were derived from a minute nodule from a bladder treated with N-methyl-N-nitrosourea (MNU); G1-200 cl-17 cells (which have low tumorigenicity) were isolated from D44c cells exposed to MNU in vitro. Neither cell line expressed TGF-alpha mRNA. The cells were cotransfected with pSV2neo and pSR alpha-rTGF-alpha. The latter plasmid contains the rat TGF-alpha cDNA under the transcriptional control of the SR alpha promoter. In the low-tumorigenicity G1-200 cl-17 cells, the expression of TGF-alpha mRNA and the subsequent synthesis of TGF-alpha protein activated epidermal growth factor receptors (EGFRs) and markedly enhanced tumorigenicity in nude mice (i.e., shortened the latency period before tumor appearance, accelerated the rate of growth, and increased the size of the tumors) as well as anchorage-independent growth in vitro. In nontumorigenic D44c cells, however, transfected TGF-alpha did not induce either anchorage-independent growth or tumorigenicity in nude mice, in spite of overexpression of EGFR mRNA and the constitutive expression of c-jun and junB mRNA. These results suggest that the increased signal transduction mediated by TGF-alpha enhanced tumorigenicity in a cell that was already tumorigenic but was not sufficient to induce tumorigenicity in a nontumorigenic cell.