We monitored the action of phospholipase A(2) (PLA(2)) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA(2) molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA(2). The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 microm(2)/s on the L-DPPC and 4.9 microm(2)/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to approximately 0.2 microm(2)/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between approximately 30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.