A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.