Fatty acid Delta(6)-desaturation, the first committed step in C(20) polyunsaturated fatty acid biosynthesis, is generally considered not to discriminate between n-3 and n-6 substrates. We previously identified higher plant species that showed preferential Delta(6)-desaturation of n-3 C(18) fatty acid substrates. A polymerase chain reaction-based approach was used to isolate 'front-end' cytochrome b(5) fusion desaturases from Primula vialii Franchet and Primula farinosa L. Functional analysis in Saccharomyces cerevisiae identified fatty acid Delta(6)-desaturases with a strong specificity for the n-3 substrate alpha-linolenic acid (18:3 Delta(9,12,15)). These results indicate that the accumulation of octadecatetraenoic acid (18:4 Delta(6,9,12,15)) in planta is due to the activity of a novel n-3-specific fatty acid Delta(6)-desaturase.