The usefulness of n-propyl-, iso-propyl-, and cyclopropylamines for the location of double bonds positions in C37-C40 alkenones after formation of imino derivatives has been evaluated. Cyclopropylamine is the best reagent for its high reaction yields, GC retention time difference between derivatives and precursor compounds, and absence of generation of byproducts. The use of this C3 amine involves higher sensitivity and ease of application than previously reported C5 amines. Examination of a large group of alkenones from cultures of Emiliania huxleyi, water particles, and recent and ancient sediments with cyclopropylamine derivatization shows that, in all cases, the double bonds were located at the same carbon atom distance from the carbonyl group, and spaced in intervals of five methylene groups either from the carbonyl or between them, e.g., at sites 7, 14, 21, and 28. This result represents a correction from previous assumptions in which double-bond positions were situated by reference to the methyl end. 4,4-Dimethyloxazoline derivatization of hexatriacontenoates showed that these compounds have also their unsaturations with seven carbon atom spacing and counting by reference to the carboxyl group. The concurrence of both series of isomers in compounds of different oxygen functionalities indicates that the precursor haptophycean algal species have a major biosynthetic pathway leading to the formation of these lipids. The data presented in this work unify the structures of the known alkenones in the present and the recent past under a common metabolic pathway.