The present study investigated developmental circadian changes in the content of 5-hydroxytryptamine (5-HT) in two structures proposed to contain pacemakers in crayfish Procambarus clarkii: the cerebral ganglion and the eyestalks. Crayfish (N=260) from three developmental stages were divided into two groups: (1) animals subjected to 12 h:12 h light:dark cycles for 10 days and (2) animals treated as described above, then exposed to 72 h of continuous dim light. Crayfish from both groups were killed at different times of day, and the cerebral ganglion and the eyestalks of each were assayed for 5-HT by reversed-phase HPLC with electrochemical detection. In all stages of development, 5-HT content (expressed as (&mgr;)g g(-)(1 )wet mass tissue) showed circadian variations in both structures analyzed; rhythms continued to free-run under constant illumination, and total 5-HT content was higher in the brain (0.581+/-0.36 (&mgr;)g g(-)(1); mean +/- s.e.m.) than in the eyestalks (0.299+/-0.15 (&mgr;)g g(-)(1)). As development advances, the percentage of the rhythm that shows periods of 24 h diminishes, while the percentage of the rhythm that shows periods of 9 to 12 h increases. This seems to indicate that pulsatile variations in 5-HT content are superimposed in a circadian component. The relationship between the 5-HT rhythm and electroretinogram and motor activity rhythms during development is discussed.