To observe the time-course of formation of sebaceous lipids in the horse, skin was pulse-labelled in vivo by intradermal injection of [1-14C]acetate and the injection sites were harvested at intervals for up to 12 days by skin punch biopsy. The distribution of radioactivity among the major neutral lipid classes and the phospholipids from these biopsies showed that, soon after pulse-labeling, the phospholipids were highly labelled followed by a long-term decrease in radioactivity. Over the same period, the low initial labelling of the dominant component, the equolides (giant ring .omega.-lactones, C32-C36), was followed by a long-term increase in radioactivity. This suggests a post-pulse transferance of radioactivity from the phospholipids to the equolides, presumably in the fatty acids. Of the phospholipid fatty acids from horse dermis, including sebaceous glands, 33% were found to contain iso-branched structures unique to horse sebaceous lipids. Of the iso-branched fatty acids, 40% were .DELTA.9-18:1 and .DELTA.9- and .DELTA.11-20:1 acids, which are structurally appropriate to be precursors for the monounsaturated equolides. These findings strengthen the hypothesis that the sebaceous phospholipids of horse skin serve as long-term intermediates in the biosynthesis of the equolides during sebaceous cell development.