Abstract: The present invention provides endothelial nitric oxide synthase (eNOS) polypeptide mutants, and polynucleotides encoding such polypeptide mutants, useful for gene therapy. In particular, the invention provides eNOS polypeptide mutants having one or more mutations in an amino acid sequence corresponding to a functional domain of a mammalian eNOS. More particularly, the invention provides eNOS polypeptide mutants having at least one mutation at a position corresponding to an amino acid residue in a calmodulin-binding site that is phosphorylated in mammalian cells, where the mutation is not an amino acid substitution to Ala or Asp in an eNOS polypeptide mutant having a single mutation that is at the phosphorylation site; and to polynucleotides encoding such polypeptide mutants. The present invention further provides prophylactic, diagnostic, and therapeutic methods of using such eNOS polypeptide mutants and polynucleotides.