Fibroblast growth factor 2 (FGF-2 or basic FGF) is associated with the cell-transformed phenotype. To clarify the function of FGF-2 in the malignancy of tumor cells, we designed experiments to express antisense RNA in a hepatoma cell line. Using FGF-2 mRNA, alternative initiations of translation at one AUG and three CUG start codons led to the synthesis of four isoforms. SK-Hep1 cells, which naturally produce the four FGF-2 proteins, were stably transfected with expression vectors that generate antisense RNAs targeted against different sites of human FGF-2 mRNA. A variable decrease of all of the isoforms of FGF-2 synthesis was observed compared with the control: the strongest inhibition was obtained with the smaller antisense targeted against AUG codon. Our results clearly demonstrated that inhibition of FGF-2 expression led to a loss of anchorage independence in soft agar. This effect was not reversed by adding exogenous FGF-2, indicating that an intracrine process of FGF-2 probably is involved in the phenotypic changes of SK-Hep1 cells. Furthermore, the inhibition of FGF-2 synthesis was correlated with a loss of tumorigenicity in nude mice. These results clearly argue for a key role of endogenous FGF-2 in transformation and tumorigenesis of the hepatoma cell line used in this study.