Abstract A cell-free particulate preparation from oyster viscera incorporated [14C]palmitate and [14C]serine into the long chain base sphingadienine. Both double bonds of the product were shown to have the trans configuration. trans-2-Hexadecenoic and trans-6-hexadecenoic acids were not effective in decreasing the incorporation of the radioactive label from palmitate, nor was label from palmitate incorporated into these two monounsaturated fatty acids. Excess sphinganine (dihydrosphingosine) and sphing-4-enine (sphingosine) added to incubation mixtures also had little effect on the level of incorporation of palmitate into the dienic base, whereas about 30% reduction was observed when 3-ketosphinganine was added. Mono- and diunsaturated 3-keto bases were produced in vitro in the absence of NADPH, and 3-keto[14C]sphinganine and 3-keto[14C]sphing-4-enine were converted in high yields to labeled 3-ketosphingadienine under these conditions. These data are consistent with a mechanism involving condensation of palmitoyl coenzyme A with serine and subsequent desaturations of the 3-ketosphinganine to form mono- and diunsaturated intermediates, followed by reduction to sphingadienine.