Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. This evidence concerns the gene NOS3 and metastatic melanoma.