In order to elucidate the effects of the different basic fibroblast growth factor (bFGF) isoforms on vascular smooth muscle, we examined aorta-derived vascular smooth muscle cells from transgenic mice expressing the human isoforms of bFGF. Four cell lines were examined from mice in which transgene expression was driven by the ubiquitous phosphoglycerate kinase promoter. Overexpression and cellular localization was confirmed by Western blot analysis in vascular smooth muscle cells from mice expressing: all four human bFGF isoforms (24, 22, 21, and 18 kDa); all three nuclear targeted isoforms (24, 22, and 21 kDa); only the 24 kDa isoform; and the only secreted/non-nuclear targeted isoform, 18 kDa. All lines showed approximate four-fold increases in bFGF expression, nuclear localization of all nuclear targeted bFGF isoforms, and cytosolic localization of only the 18 kDa bFGF. Measurement of [³H]thymidine incorporation into quiescent cells stimulated with increasing concentrations of serum, showed increased DNA synthesis in cell lines expressing any bFGF isoform when compared to non-transgenic control cells, and a further increase in DNA synthesis in cells expressing the nuclear targeted isoforms (24, 22, and 21 kDa) over the 18 kDa bFGF expressing cell line at any concentration of serum. All cells showed equal label incorporation when stimulated with 10 ng/ml of platelet-derived growth factor confirming an equal potential for DNA synthesis. Neutralizing the bFGF antibody markedly decreased serum-stimulated DNA synthesis, but only in the cell lines overexpressing the secreted/non-nuclear targeted 18 kDa isoform. These results suggest amplification of DNA synthesis through synergistic intracrine and autocrine effects of the nuclear targeted and non-nuclear targeted bFGF isoforms in vascular smooth muscle cells.