Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.