In an effort to determine the driving force of the diurnal variation of serotonin (5-HT) in the brain, over 500 Syrian hamsters were exposed to long photoperiods (LD17:7; LD), short photoperiods (LD8:16; SD), constant dark (DD) or constant light (LL) for 12 weeks. Hamsters exposed to LD or SD were sacrificed at 3 h intervals; those in constant conditions were sacrificed around the clock. The circadian time (CT) of tissue collection, in the animals in constant conditions, was determined from the onset of locomotor activity (defined as CT12; the beginning of the subjective night). Serotonin content was determined by HPLC with electrochemical detection in the medial basal hypothalamus (MBH); anterior hypothalamus (AH) and olfactory bulbs (OB). In LD and SD, 5-HT levels displayed significant diurnal variation in the MBH, AH and OB (ANOVA; P less than 0.001). The sine waves of the 5-HT rhythm in these conditions were similar in amplitude and phase with relation to lights on (i.e. high 5-HT content during the day and low content at night, with a sharp rise occurring just after lights on). This variation, however, was not apparent in animals exposed to DD or LL; 5-HT content did not display a significant diurnal oscillation. Since 5-HT failed to oscillate in the absence of environmental time cues, the rhythm is likely driven by the environment and not an internal circadian clock.