The role of neural activity in the long-term regulation of tyrosine hydroxylase in peripheral adrenergic neurons had been studied by electrically stimulating the preganglionic nerves innervating the superior cervical ganglion. Tyrosin hydroxylase activity was elevated maximally 3 days after stimulation. The magnitude of the increase varied from 25 to 120% depending on the frequency and duration of the stimulation. Dopamine-beta-hydroxylase activity was also increased after preganglionic nerve stimulation, but there was no change in dopa decarboxylase activity or in the ganglion protein content. The increase in tyrosine hydroxylase activity is paralleled by a comparable increase in the amount of immunoreactive tyrosine hydroxylase in the ganglia. This increase in enzyme activity could be prevented by pretreating animals with the ganglion-blocking drugs hexamethonium or chlorisondamine but was unaffected by administration of atropine or dihydroergotamine. Antidromic stimulation did not mimic the effects of orthodromic stimulation indicating that an increase in the firing rate of the neurons was not a sufficient condition for the long-term elevation of tyrosine hydroxylase activity. These studies demonstrate that periods of increased synaptic stimulation can alter the protein composition of sympathetic neurons, increasing the specific activity of the key enzyme involved in their transmitter biosynthesis.