The type of dietary fat affected the production of cAMP by cultured thyroid cells incubated with mouse and rat sera. Greater amounts of cAMP were produced with serum from mice fed 30% rac-1(3)-palmitoyl glycerol and 4% safflower oil (PG + SO) than with serum from mice fed 30% rac-1(3)-palmitoyl glycerol (PG). The serum from mice fed PG + SO gave a response similar to that with calf serum. Sera were separated into lipoprotein and aqueous fractions by centrifugation. A combination of both the lipoprotein and aqueous fractions of serum from mice fed PG + SO was required for the increased response. Proteolysis of the aqueous fraction of the serum from either mice fed PG or rats fed 30% hydrogenated coconut oil (HCO) reduced the amount of cAMP produced by the thyroid cells. However, the same effect was not seen with aqueous fractions of calf serum or serum from mice fed PG + SO or rats fed HCO plus 5% corn oil. These findings suggest that there are at least three factors in serum capable of regulating thyroid cell metabolism that are controlled by the type of fat fed the animal.