1. Reserpine in vitro (10(-5)M) caused a profound inhibition (>85%) of the formation of both (14)C-catecholamine ((14)C-CA) and (14)C-dihydroxyphenylalanine ((14)C-DOPA) (in the presence of the amino acid decarboxylase inhibitor brocresine) from (14)C-tyrosine in guinea-pig vas deferens. The magnitude of the inhibition was similar for both (14)C-CA and (14)C-DOPA suggesting that the inhibition occurred primarily at the tyrosine hydroxylase step.2. One hour after in vivo treatment with reserpine (1 mg/kg) when tissue stores of noradrenaline (NA) were depleted by 50%, there was a significant inhibition of the formation of (14)C-DOPA. Twenty-four hours after such treatment, when endogenous NA could no longer be detected, synthesis of (14)C-DOPA was indistinguishable from untreated controls. However a 45% inhibition of (14)C-DOPA synthesis from (14)C-tyrosine could be produced in tissues which had been depleted of NA for 24 h or 48 h by the addition of reserpine, 10(-5)M, to the incubation medium.3. Addition of pteridine cofactor, 2-amino-6,7,-dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine, to the incubation medium in a concentration of 5 x 10(-3)M enhanced the formation of both (14)C-CA and (14)C-DOPA from (14)C-tyrosine in guinea-pig vas deferens. In 52 mM KCl Krebs-Henseleit medium (14)C-CA formation increased from 2.58+/-0.20 (nmol/g)/h to 6.35+/-0.47 (nmol/g)/h whilst (14)C-DOPA formation increased from 5.04+/-0.88 (nmol/g)/h to 11.29+/-0.59 (nmol/g)/h.4. Pteridine cofactor (5 x 10(-3)M) did not reverse the inhibition of (14)C-DOPA formation seen with reserpine (10(-5)M) in previously untreated tissues or in vasa deferentia from animals pretreated with reserpine 1 mg/kg for 24 hours. However, the inhibition did disappear in the presence of pteridine cofactor when treatment with reserpine was prolonged to 48 h and included two doses of reserpine of 2 mg/kg.5. Tyramine (5.8 x 10(-5)M) and bretylium (10(-5)M) in vitro inhibited the formation of (14)C-CA and (14)C-DOPA from (14)C-tyrosine to the same extent in guinea-pig vas deferens again indicating that their major site of action is on tyrosine hydroxylase. The inhibitory effects were reversed by pteridine cofactor.6. Synthesis of (14)C-NA from (14)C-tyrosine in calf splenic nerve was not increased by incubating the tissue in 52 mM KCl-Krebs-Henseleit solution.