Sleep-promoting factor (factor S) was extracted, partially purified, and concentrated from cerebrospinal fluid and from acid-acetone extracts of brain stem anc cortex of sleep-deprived goats and sheep. 2. Solutes greater than 500 daltons were largely removed by serial ultrafiltrations through molecular sieves (Amicon membranes UM10 and UM05); solutes less than 350 daltons were largely eliminated by gel filtration through Sephadex G10 columns. Sleep-promoting activity was found in a fraction eluted prior to [14C] sucrose marker. 3. Concentrated fraction were infused intraventricularly in rats (0.1 ml in 30 min just prior to 12-h dark cycle) and in rabbits (0.3 ml in 90 min in morning). Sleep-promoting activity was assayed by decrease in nocturnal locomotor activity of rats and by duration and amplitude of slow-wave cortical EEG in rabbits.