Thin-layer chromatography with a flame ionization detector (TLC-FID) was used for monitoring the production of structured phospholipids (ML type: L, long-chain fatty acids; M, medium-chain fatty acids) by enzyme-catalyzed acidolysis between soybean phosphatidylcholine (PC) and caprylic acid. It was found that the structured PC fractionated into two to three distinct bands on both plate thin-layer chromatography (TLC) and Chromarod TLC. These three bands represented PC of the LL type, ML type, and MM type, respectively. The TLC-FID method was applied in the present study to examine the influence of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio (caprylic acid/PC, mol/mol) on formation of ML-type PC in a batch reactor with Thermomyces lanuginosa lipase as the catalyst. The formation of ML-type PC was dependent on all parameters examined except for the substrate ratio. The ML-type PC content increased with increasing enzyme dosage, reaction temperature, solvent amount, and reaction time. The substrate ratio had no significant effect on the formation of ML-type PC within the tested range (3-15 mol/mol). The formation of MM-type PC was observed in some experiments, indicating that acyl migration is taking place during reaction since the lipase is claimed to be 1,3-specific. The TLC-FID method offers a simple and cheap technique for elucidation of product and byproduct formation during enzyme-catalyzed reactions for production of phospholipids containing mixtures of long- and medium-chain fatty acids.