Two types of VEGFR-1 receptors have been characterized: a full-length transmembrane receptor and a truncated extracellular soluble isoform (sVEGFR-1). We report here the characterization, in normal and cancer cells, of a new family of intracellular isoforms of VEGFR-1 resulting from alternative initiation of transcription in intronic sequences of the gene. While the classical isoforms of VEGFR-1 were barely detectable in MDA-MB-231 breast cancer cells, one of the intracellular isoforms transcribed from intron 21 (i21VEGFR-1) was the main isoform expressed in these cells. The new transcript encodes for a protein that contains only the phosphotransferase domain and the carboxyterminal tail of VEGFR-1. Treatment of MDA-MB-231 cells with siRNA specific for the tyrosine domain of VEGFR-1 suppressed the expression of i21VEGFR-1, downregulated phosphorylation of Src at tyrosine 418, and reduced markedly the invasion capacity of these cells in vitro. Accordingly, overexpression of transfected i21VEGFR-1 in MDA-MB-231 cells upregulated the active form of Src and increased invasiveness of MDA-MB-231 cells. The expression of i21VEGFR-1 in MDA-MB-231 cells was inhibited by retinoic acid. Both, activation of Src and downregulation by retinoic acid, have been reported in other intracellular members of the Fms/Kit/PDGFR family of tyrosine kinases, particularly in the intracellular isoform of c-kit, analogous structurally to i21VEGFR-1 and frequently expressed in cancer cells. J. Cell. Biochem. 110: 732–742, 2010. © 2010 Wiley-Liss, Inc.