The accumulation of sleep regulatory substances (SRS) in cerebrospinal fluid (CSF) during prolonged wakefulness provides very strong support for the hypothesis that sleep is regulated, in part, by humoral agents.reviewed, 1–4 Many substances can affect sleep, although only a handful of humoral agents are strongly implicated in sleep regulation. The list includes tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), growth hormone releasing hormone (GHRH), prostaglandin D2 and adenosine for nonrapid eye movement sleep (NREMS), and vasoactive intestinal peptide and prolactin for REMS.reviewed,1,4,5 All putative SRSs thus far identified have other biological activities not directly tied to sleep. For example, IL-1 and some prostaglandins are pyrogenic, yet body temperature decreases upon entry into NREMS. 6–8 Similar problems of specificity confront the humoral and neuronal regulation of all physiological functions. Any manipulation that alters sleep can also alter other physiological parameters, e.g., sleep deprivation is associated with changes in body temperature, food intake, and endocrine function. It is therefore our opinion that independent approaches affecting sleep should be used to determine sleep-specific changes in either SRSs or in neuronal networks. Thus, if one is interested in determining sleep-specific localized changes in an SRS mRNA, only changes mutually induced by independent sleep-altering methods can be considered candidates for specific SRS sleep mechanisms.