We analyzed the molecular mechanism controlling ω-3 fatty acid desaturases during seed germination and leaf development in soybean. During germination, soybean seeds were characterized by a high 18:2(Δ9,12) level (more than 50%) and reduced 18:3(Δ9,12,15) content (10%). Interestingly, transcripts from all endoplasmic reticulum (GmFAD3A and GmFAD3B) and plastidial (GmFAD7-1/GmFAD7-2 or GmFAD8-1/GmFAD8-2) desaturase genes were detected during seed germination. Upon germination, soybean trifoliate leaf development was accompanied by an increase in linolenic acid (18:3(Δ9,12,15)). Our data showed that transcripts corresponding to the endoplasmic reticulum ω-3 desaturases GmFAD3A and GmFAD3B decreased with leaf development. No changes in the expression profile of the plastidial ω-3 desaturases GmFAD7-1 and GmFAD7-2 genes were detected. On the contrary, GmFAD8-2 transcript levels increased while GmFAD8-1 transcripts decreased during leaf development. Given this expression profile, our data suggested the existence of a temporal regulatory mechanism controlling ω-3 desaturases during leaf development in which the endoplasmic reticulum ω-3 desaturases would be more important in young leaves while plastidial ω-3 desaturases might contribute to 18:3(Δ9,12,15) production in mature leaves. Photosynthetic cell cultures showed 18:3(Δ9,12,15) levels similar to those from leaves. No changes in the 18:3(Δ9,12,15) content or expression of the ω-3 desaturase genes were detected along the cell culture cycle. A comparison of our data with those available in Arabidopsis or wheat suggested that the regulatory mechanism controlling the expression and activity of both endoplasmic reticulum and plastidial desaturases during leaf development might differ among plant species.