Monolayer cultures of superior cervical ganglia free of support cells were maintained in the presence of 100 ng/ml 7S-NGF for 4 days. The concentration of NGF was then changed to between 50 and 400 ng/ml and cultures continued for an additional 7 days. Tetrahydrobiopterin (BH4) content, GTP cyclohydrolase (GTPCH) enzyme activity and mRNA levels were then determined. All three of these measures were found to be elevated between 2- to 4-fold by treatment with increasing concentrations of NGF. Tyrosine hydroxylase (TH) enzyme activity and mRNA levels were increased from 8 to 13-fold by these same treatments. These results indicate that the content of BH4 within sympathetic neurons can be regulated by NGF receptor-mediated changes in GTPCH gene expression. Moreover, concomitant increases in TH enzyme activity and BH4 content demonstrate a coordinated regulation by NGF of this enzyme and its essential cofactor.