Specific phospholipase A(2) (PLA(2)) adsorption studies were performed on monolayers of D-dipalmitoyl-phosphatidylcholine (D-DPPC) and of an ether-ester D-1-O-hexadecyl-2-stearoyl-phosphatidylcholine (D-HSPC). In order to separate interfacial recognition from subsequent lipid cleavage, PLA(2)-resistant D-enantiomers were utilized for the investigations. Snake venom (N. naja naja and Crotalos atrox) PLA(2), which hydrolyzes the sn-2 ester bond of L-phospholipids, was used. Fluorescence microscopy, film-balance pressure-area isotherms, and grazing incidence X-ray diffraction (GIXD) experiments were carried out. Fluorescence microscopy studies show that the enzyme accumulates preferentially at the liquid-expanded/condensed interface. At low surface pressure enzyme penetration into the monolayer is observed, whereas at high pressures the area per molecule is reduced upon specific adsorption. Monolayer structure, as determined by GIXD, is greatly affected by adsorption of PLA(2). The tilt angle of the aliphatic chains of the monolayer becomes drastically reduced due to an enzyme-induced increase of the lipid packing efficiency. The unspecific adsorption of serum albumin to a D-DPPC monolayer does not change the monolayer structure. The structural changes, caused by PLA(2) adsorption on D-enantiomer monolayers, are related to the chemical structure of the lipid molecules. Therefore, a relation between structure change and hydrolysis efficiency of PLA(2) on the respective L-enantiomer monolayers can be assumed.