The in vitro capacity of sympathetic superior cervical ganglia (SCG) to take up [3H]choline from the extracellular medium, to synthesize acetylcholine from [3H]choline, and to release [3H]acetylcholine in response to a high K+ concentration, were examined in rats throughout a 24-h cycle. Both the release of [3H]acetylcholine and the synthesis of [3H]acetylcholine from [3H]choline exhibited significant diurnal variations, showing maxima during the first half of the night. After these maxima, nocturnal acetylcholine release and synthesis decayed to daytime levels and remained low until the end of the night. [3H]Choline uptake by rat SCG did not vary significantly throughout a 24-h period. A 1.5-h exposure of rats to darkness at the 5th hour of light phase of the daily photoperiod did not change significantly any parameter studied. A 20-min, 5-Hz, electrical stimulation of the preganglionic trunk of SCG excised from rats at noon increased significantly subsequent K(+)-induced [3H]acetylcholine release but did not change [3H]acetylcholine synthesis. In decentralized SCG of rats subjected to a unilateral SCG decentralization and a contralateral sham-operation 7 days earlier, [3H]acetylcholine release and synthesis were highly reduced or abolished at the decentralized side, while [3H]choline uptake remained unaltered. The present results suggest that an activation of preganglionic rat SCG neurons takes place during the first half of the scotophase.