Fibroblast growth factor-1 (FGF-1) is a potent mitogen for mesoderm- and neuroectoderm-derived cell types in vitro. However, a mutant FGF-1 with deletion in its nuclear localization sequence (NLS, residues 21-27) is not mitogenic in vitro. We demonstrated that synthetic peptides containing this NLS were able to stimulate DNA synthesis in a FGF receptor-independent manner after they were delivered into living NIH 3T3 cells by a cell-permeable peptide import technique. The stimulation of maximal DNA synthesis by these peptides required the presence of peptides during the entire G1 phase of the cell cycle. The mitogenic effect was specific for the NLS of FGF-1 because a peptide with double point mutations at lysine residues was inactive in stimulating DNA synthesis. Our results suggest that the NLS plays an important role in the mitogenic pathway initiated by exogenous FGF-1 by its direct involvement in the nuclear transport and signaling of internalized FGF-1.