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Omega-3 long chain polyunsaturated fatty acids (&#969;3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5&#969;3) and docosahexaenoic acid (DHA; 22:6&#969;3) are important fatty acids for human health. These &#969;3 LC-PUFAs are produced from their &#969;3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from &#969;6 LC-PUFA by omega-3 desaturases (&#969;3Ds). Here, we have investigated eight &#969;3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 &#969;6 PUFA substrates by transiently expressing them in <i>Nicotiana benthamiana</i> leaves. Our results showed hitherto unreported activity of many of the &#969;3Ds on &#969;6 LC-PUFA substrates leading to their conversion to &#969;3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.