The intracellular thyroid hormone (TH) availability is influenced by different metabolic pathways. We investigated the relationship between tissue and plasma TH levels as well as the correlation with changes of deiodination and sulfation during chicken embryonic development. From day 14 until day 19, T3 remains unchanged in liver and kidney in spite of increasing plasma T4 and T3 levels and a slightly increased T4 availability in these tissues. During this period, the T3 breakdown capacity by type III deiodinase (D3) is high in liver but low in kidney. The TH inactivation capacity of type I deiodinase (D1), with production of inactive rT3 instead of T3, in kidney seems to be potentiated by the sulfation pathway. A sharp rise in T3 and T4 is detected in all tissues examined when the embryo switches to lung respiration. The same day, T4 content in liver is sharply enhanced and sulfation activity is decreased. So, T4 availability in liver is increased while a declined D3 activity allows for the accumulation of hepatic T3. The increase in renal T3 and T4 are more closely related to plasma TH profiles and a lack of correlation with the changes in renal D1 and D3 activity suggests that T4 and T3 content in this organ is strongly dependent on direct uptake from the blood. Despite much lower T4 levels, T3 levels in brain are in the same range as in liver and kidney and intracellular T3 even exceeds the T4 levels towards the end of development. The rise in TH content coincides with a drop in D3 activity, low sulfation activity and an increased T3 production capacity via type II deiodinase (D2). In conclusion, the current study describes the dynamics of intracellular TH concentrations in liver, kidney, and brain during chicken development and investigates their relationship with circulating TH levels and changes of deiodinases and sulfotransferases. The clear differences in intracellular TH profiles among the different tissues demonstrate that circulating levels are not necessarily representative for the local TH changes. Some of the changes in intracellular TH availability can be linked to changes in local deiodination and sulfation capacities, but the importance of these enzyme systems in relation to other factors, such as hormone uptake, differs between liver, kidney, and brain.