Notes: —Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P 〈 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide.In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [3H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [3H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP.The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm-potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP.Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.