In the present study, tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was transfected into cultured fibroblasts and the production of L-3,4-dihydroxyphenylalanine (L-DOPA) was determined. Type 2 TH cDNA was transfected into fibroblasts (NRK-49F) derived from the normal rat kidney, and the expression of the TH minigene was screened by immunocytochemical staining and immunoblotting analysis with TH antiserum. Several clones of the NRK transfectants that produce TH molecules were obtained. The expressed TH molecules showed high enzyme activity in a complete assay system in vitro. However, L-DOPA was not detected in the cultured cells due to the possible absence of de novo synthesis of (6R)-L-erythro-tetrahydrobiopterin (BH4) in these cells. When BH4 was added to the medium, a large amount of L-DOPA was detected not only in the cells but also in the medium. These findings may aid in regulating the amount of L-DOPA secretion from cells after they are transplanted into the brain.