The relationship between serum cholesterol concentrations and serotonin synthesis rate in brain was examined in Mongolian gerbils chronically fed diets containing 20% fat (safflower oil, beef tallow or butterfat) with or without added cholesterol (0.5%, dry weight). After 22 days on these diets, circulating cholesterol concentrations ranged between approximately 1.5 and approximately 20 mumol/ml. Despite this enormous range, in vivo tryptophan hydroxylation rate, and serotonin and 5-hydroxyindoleacetic acid concentrations in cerebral cortex, hypothalamus and brainstem did not differ significantly among the diet groups. Tryptophan concentrations in serum and brain were also unaffected. These results do not support the hypothesis that the link between depression, suicide and violent deaths and below-normal or reduced serum cholesterol concentrations in humans involves an alteration in serotonin synthesis and/or release by brain neurons.