In Rhizobium leguminosarum the nodABC and nod-FEL operons are involved in the production of lipooligosaccharide signals, which mediate host specificity. A nodE-determined highly unsaturated C18:4 fatty acid (trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule primordia (Spaink, H. P., Sheeley, D. M., van Brussel, A. A. N., Glushka, J., York, W.S., Tak, T., Geiger, O., Kennedy, E. P., Reinhold, V. N., and Lugtenberg, B. J. J. (1991) Nature 354, 125-130) and preinfection thread structures (van Brussel, A. A. N., Bakhuizen, R., van Spronsen, P. C., Spaink, H. P., Tak, T., Lugtenberg, B. J. J., and Kijne, J. W. (1992) Science 257, 70-72) on the host plant Vicia sativa. We presently focus on the question of how these lipo-oligosaccharide signals are synthesized in Rhizobium. Here we show that after the induction of the nodFE genes, even in the absence of the nodABC genes, the trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid, which has a characteristic absorbance maximum of 303 nm, is synthesized; this shows that the biosynthesis of the unusual fatty acid is not dependent on the synthesis of the lipooligosaccharides. This finding also suggests that the biosynthesis of the unusual fatty acid is completed before it is linked to the sugar backbone of the lipooligosaccharide. In an attempt to identify the lipid fraction with which the unusual C18:4 fatty acid is associated, we found that it is linked to the sn-2 position of the phospholipids. Even when lipooligosaccharide signals are produced in a wild type Rhizobium cell, a fraction of the unusual fatty acid is still bound to all major phospholipids. These findings offer interesting possibilities. 1) The phospholipids might be biosynthetic intermediates for the synthesis of lipooligosaccharide signals, and 2) phospholipids, containing nodFE-derived fatty acids, might have a signal function of their own.