The effect of low (10°C) and high (30°C) temperature on in vivo oleate desaturation has been studied in developing sunflower (Helianthus annuus L.) seeds under conditions of different oxygen availability (capitulum, detached achenes or peeled seeds). In seeds remaining in the capitulum, only a part of the oleate newly synthesized at high temperature was desaturated to linoleate, whereas more oleate than that synthesized de novo was desaturated at low temperature. Achenes were only able to significantly desaturate oleate at low temperatures. In contrast, oleate desaturation was detected in peeled seeds incubated at low and high temperatures, showing the highest rate at 20°C. Hull removing dramatically increased the activity of the microsomal oleate desaturase (FAD2, EC 220.127.116.11) at all studied temperatures, although a long-term inactivation of the enzyme was observed at high temperatures. Low oxygen concentration (1–2%) obtained by respiration of peeled seeds incubated in sealed vials, brought about the inactivation of the enzyme. All these data suggest that temperature regulates oleate desaturation controlling the amount of oleate and the FAD2 activity. In addition, this enzyme seems to be also regulated by the availability of oxygen, which is affected inside the achene by its diffusion through the hull, and the competition with respiration, both factors being temperature-dependent.