The localization of immunoreactive T3 was investigated in dog and chick thyroid glands either fixed in Bouin's solution or freeze-dried and fixed with paraformaldehyde vapor, and compared with localization of 19S-thyroglobulin. Freeze-drying followed by paraformaldehyde vapor was better for the demonstration of T3 than Bouin's solution; it gave a much stronger immunoreactivity for T3. This fixative was also excellent for the demonstration of thyroglobulin. The immunoreactive T3 was detected only in the colloid and was never observed in the follicular cells, although immunoreactive thyroglobulin was present not only in the colloid but also in the follicular cells. Subsequently, in dog fetuses and chick embryos the appearance and development of immunoreactive T3 were studied. At 40 days of gestation in dog fetuses and at 9 days of egg incubation in chick embryos, immunoreactive T3 was found in the colloid of primordial follicles coinciding with the formation of the colloid. The ability of embryonic thyroid glands to synthesize T3 seems to be linked to the organization of follicles. With progressing development, the follicles stored more colloid and immunoreactive T3 within the follicular lumina. Concentrations of immunoreactive T3 in thyroids from chickens at various stages of development were also studied by radioimmunoassay. The T3 concentration was first detected at 9 days of incubation and gradually increased with embryo age; it was related to the amounts of colloid stored in the follicles.