Ligand activation of fibroblast growth factor receptor-1 (FGFR-1) induces an angiogenic response following activation of multiple intracellular signaling substrates, including the Src family of nonreceptor tyrosine kinases (SFK). However, the direct association between FGFR-1 and SFK and the involvement of SFK in FGFR-1-dependent cell proliferation have been controversial. Structural variants of FGFR-1 are generated by alternative splicing which results in two major isoforms, containing either three (FGFR-1alpha) or two (FGFR-1beta) immunoglobulin-like domains in the extracellular region. To determine whether alternatively spliced FGFR-1 isoforms differentially activate SFK, we have examined FGF receptor-negative endothelial cells stably transfected with human cDNA encoding either FGFR-1alpha or FGFR-1beta. Transient activation of c-YES, the predominant SFK expressed in these endothelial cells, was restricted to FGFR-1beta transfectants following exposure to acidic fibroblast growth factor (FGF-1). Co-immunoprecipitation studies revealed that c-YES directly associated with FGFR-1beta. The Src homology (SH)2 domain (and not the SH3 domain) of c-YES was able to recognize tyrosine phosphorylated FGFR-1beta. FGFR-1beta-specific activation of c-YES was accompanied by its association with and activation of cortactin. FGF-1 treatment of both FGFR-1alpha and FGFR-1beta transfectants induced SFK-independent cellular proliferation and growth in low density cultures. At high density, under both anchorage-dependent and -independent conditions, FGF-1 failed to induce proliferation and growth of FGFR-1alpha transfectants. In contrast, FGF-1 induced proliferation, growth, and formation of cord-like structures in high density cultures of FGFR-1beta transfectants in an SFK-dependent manner. In vitro cord formation on Matrigel was restricted to FGFR-1beta transfectants in an SFK-dependent manner. Formation of vascular structures in vivo was limited to endothelial cells transfected with FGFR-1beta. Collectively, these results emphasize the roles of alternatively spliced FGFR-1 structural isoforms and activation of SFK as modulators of endothelial cell growth during the formation of neovascular structures.