Results with the "sulfo-phospho-vanillin" reaction, much used for determining total serum lipids, have been favorably compared with those for the gravimetric method. We investigated the basic chemistry of the reaction and determined the reactivity of this single reagent with various lipids. Our results suggest that: (a) The reaction requires a carbon-carbon double bond. (b) Concentrated sulfuric acid reacts with unsaturated lipids in the initial step to form a carbonium ion. (c) Phosphoric acid reacts with vanillin to produce a phosphate ester, with a resulting increase in the reactivity of the carbonyl group. (d) The carbonium ion reacts with the carbonyl group of phosphovanillin to form a colored compound, which is stabilized by resonance. (e) Unsaturated compounds with more than one double bond react, but reaction may vary with steric hindrance. (f) The method is reasonably precise, but its accuracy depends primarily on the reference standard used.