The role of HIF-3α in response to intermittent hypoxia and physical exercise was investigated in Fisher rats using reverse transcription, real-time PCR and RNA interference. Under acute hypoxia (12% O(2), 2h), the level of HIF-1α, HIF-2α, and HIF-1β mRNA did not significantly change in the heart, lung, kidney and muscle tissues, but HIF-3α mRNA expression was strongly elevated in all tissues investigated. Five weeks of intermittent hypoxic training (IHT) led to an increase of HIF-3α mRNA in all studied tissues, but under the influence of acute hypoxia after IHT, the expression of HIF-3α mRNA did not increase in all tissues excluding skeletal muscle. Thus, IHT reduced the effect of acute hypoxia on HIF-3α mRNA expression in the heart, lung, and kidney. The expression of HIF-3α in skeletal muscle at endurance (swimming) training (ET) in combination with IHT was 6.2 times lower compared to the group with ET but without IHT and 3.3 times lower (P<0.05) compared to untrained control. After swimming training with IHT, rats demonstrated the highest level of physical endurance. RNA interference of HIF-3α significantly decreased the level of HIF-3α mRNA in both muscles investigated: by 2.6 times in m. soleus (P<0.03) and by 2.1 times in m. gastrocnemius (P<0.05) and elevated the physical endurance of rats by 50% (P<0.05) compared to control. Thus, the HIF-3α subunit is an essential member of hypoxic response. It plays a negative role in the adaptation to hypoxia, because the inhibition of HIF-3α expression leads to an increase in physical endurance.