The effect of lipid hydrophobic chain lengths on adsorption of glucose oxidase (GOx) into phospholipid monolayers has been investigated. The results show that at low initial surface pressures, penetration of GOx depended both on hydrophobic interactions and on the orientation of lipid chains with respect to the interface plane. Compared with dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC), dibehenoyl-phosphatidylcholine (DBPC) favored enzyme adsorption at low surface pressures. This was attributed to its long hydrophobic chains and to their vertical orientation. At higher initial surface pressures, the fluidity and packing of these monolayers appeared to play a key role in the penetration process. The presence of a double bond in the hydrocarbon chains enhanced enzyme adsorption, and this effect was independent of the initial surface pressure of lipid monolayers.