The early steps of spiroacetal biosynthesis in the fruit fly Bactrocera cacuminata (Solanum fly) have been investigated using a series of deuterium-labeled, oxygenated fatty acid like compounds. These potential spiroacetal precursors were administered to male flies, and their volatile emissions were analyzed for specific deuterium incorporation by GC/MS. This has allowed the order of early oxidative events in the biosynthetic pathway to be determined. Together with the already well-established later steps, the results of these in vivo investigations have allowed essentially the complete delineation of the spiroacetal biosynthetic pathway, beginning from products of primary metabolism. A fatty acid equivalent undergoes a series of enzyme-mediated oxidations leading to a trioxygenated fatty acid like species that includes a vicinal diol. This moiety then undergoes enzyme-mediated oxidative carbon-carbon bond cleavage as the key step to generate the C9 unit of the final spiroacetal. This is the first time such an oxidative transformation has been reported in insects. A final hydroxylation step is followed by spontaneous spiro-cyclization. This distinct pathway adds further to the complexity and diversity of biosynthetic pathways to spiroacetals.