A role for 5-hydroxytryptamine (5-HT) or serotonin in sleep has been known for decades but was challenged by recent papers which concluded that the apparent sleep phenotype was secondary to defective thermoregulation. Those studies used mice lacking serotonergic neurons resulting from the loss of function mutations in the gene encoding the LIM homeobox transcription factor 1 (Lmx1b). Here we show that, while Lmx1b mutants failed to keep the physiological body temperature, they exhibited more activities at the room and elevated temperatures. More importantly, we used mice deficient in the gene encoding tryptophan hydroxylase 2 (Tph2) which could not synthesize 5-HT in the brain. Tph2 mutants were capable of thermoregulation and keeping physiological body temperature when the environmental temperature was reduced and exhibited significantly more activities at both the room and elevated temperatures. Electroencephalographic (EEG) recording also showed decreased sleep in Tph2 deficient mice. Our results indicate that 5-HT is important for sleep regulation but not thermoregulation.