1-O-Octadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAP) belonging to the class of single-chained ether phospholipids is widely known from its essential biological activities. There is a growing body of evidence that some significant aspects of PAP actions are connected with its capability to direct intercalation into biomembranes' environment. Although this mechanism is of great importance in the perspective of understanding PAP implications in various physiological processes, in the literature, there is a lack of studies devoted to this subject. It is still unknown which is the exact influence of membrane composition, molecular organization, and its other properties on the PAF impact on cells and tissues. Unfortunately, the biological studies carried out on cell cultures do not provide satisfactory results, mainly because of the complexity of natural systems. In order to obtain insight into the behavior of PAP in a lipid environment at the molecular level, the application of appropriate model systems is required. Among them, Langmuir monolayers are very often applied as a simple but very efficient platform for studies of the interactions between membrane lipids. In the present paper, special attention is focused on the issue concerning the interactions between PAP and two representatives of membrane components occurring mainly in the outer leaflet of natural bilayers, namely, cholesterol and DPPC. The application of Langmuir monolayers enabled us to construct the effective model mimicking the exogenous incorporation of PAP into membrane environment. On the basis of the obtained results, a thorough discussion was carried out and the conclusions derived from the traditional thermodynamic analysis were confronted with microscopic analysis of surface domains and the GIXD results. The selection of experimental techniques enables us to obtain information regarding the miscibility and interactions in the binary mixed films as well as the molecular organization of film-forming molecules on water surface. The experiments revealed that the addition of the investigated single-chained ether phospholipid into both cholesterol and DPPC monolayers causes a considerable decrease of monolayer condensation. On the basis of thermodynamic analysis, it was found that PAP mixes and consequently interacts strongly with cholesterol, whereas its interactions with DPPC are thermodynamically unfavorable. Differences between the PAP influence on cholesterol and DPPC monolayer found its corroboration in the results obtained with the GIXD technique. Namely, the monolayer of DPPC can incorporate more PAP than the model membrane containing cholesterol. The obtained results indicate that short chained sn-2 ether phospholipid is able to modify model membrane properties in a concentration-dependent way.