This study deals with the interplay between the interfacial structure of lipid liquid crystalline (1c) substrates and the lipolysis rate. Thermomyces (formerly Humicola) lanuginosa lipase (TLL) was added to lamellar (L-alpha), reversed bicontinuous cubic (C), and reversed hexagonal (H-II)1c phases, based on monoolein (MO), MO-sodium oleate (NaO), MO-oleic acid (OA), and MO-diolein (DO) with water. The changes in self-assembled structure and lipid composition during lipolytic processes were followed by polarizing microscopy, small-angle X-ray diffraction, and high-performance liquid chromatography (HPLC). Indeed, the observed changes in self-assembled structures could be predicted from either the MO-OA-(H2O)-H-2 ternary phase diagram, where the lipolysis gives rise to a transition of C --> H-II --> micellar cubic (C-mic) --> reversed micellar phase + dispersion, or the MO-NaO-(H2O)-H-2 ternary phase-diagram, where the corresponding sequence is L-alpha --> H-I. These observations are discussed in terms of the degree of protonation of the fatty acid. The specific activity of TLL on the C-D and OA-H-II samples as determined from the lipolysis rate was found to be the similar under. the employed experimental conditions. The HPLC data showed that the ratio between the substrate (MO/DO) and final product (OA) approached about the same values regardless of the initial substrate composition and structure.