Background. Human inducible nitric oxide synthase (iNOS) gene transfer inhibits myointimal hyperplasia in vitro. However, unstimulated vascular smooth muscle cells (SMC) do not synthesize tetrahydrobiopterin (BH4), an essential cofactor for iNOS, which may be an obstacle to successful vascular iNOS gene therapy. We investigated the capacity of gene transfer of guanosine triphosphate (GTP) cyclohydrolase I (GTPCH), the rate-limiting enzyme for BH4 biosynthesis, to supply cofactor for iNOS activity. Methods. A human GTPCH expression plasmid (pCIS-GTPCH) was transfected into rat aortic SMC (RAOSMC) and BH4-deficient NIH3T3 cells engineered to stably express human iNOS (3T3-iNOS). GTPCH activity and intracellular biopterins were assessed as a measure of successful transfection, and the capacity of GTPCH to reconstitute iNOS activity was used to determine whether BH4 was made available to the iNOS protein. Results. The pCIS-GTPCH-transfected 3T3 cells had demonstrable GTPCH activity as compared with control cells (169.3 ± 6.6 pmol/hr/mg versus 0, p < 0.001). Intracellular biopterin levels were also increased in transfected 3T3 and SMC (60.6 ± 2.6 and 101.7 ± 28.3 pmol/mg, respectively, versus less than 4 in control cells). GTPCH reconstituted near-maximal iNOS activity in 3T3-iNOS cells despite a gene transfer efficiency of less than 1%. GTPCH and iNOS enzymes did not have to coexist in the same cell for the synthesized BH4 to support iNOS activity. Conclusions. GTPCH gene transfer reconstitutes iNOS activity in BH4-deficient cells despite poor transfer efficiency. GTPCH can deliver a cofactor to targeted cells even if it is synthesized in neighboring cells, and may be a means to concurrently deliver BH4 with iNOS in vivo.