To investigate the effect of saline on miscibility, phase, and conformational transitions in binary mixtures of a succinyl-phosphoethanolamine bearing C-16 aliphatic chains, DPPE-succinyl, and a poly(ethylene glycol) (PEG)-phospholipid conjugate with a PEG molecular weight of 2000, DPPE-PEG2000, we have compared the properties of monolayers spread on water and on phosphate buffered saline (PBS). A comparative analysis of monolayer surface pressure, surface potential, compressibility, and epifluorescence microscopy data has revealed that spreading on PBS induces unfavorable interactions between the two phospholipids, which stabilizes immiscible phases in mixed monolayers. Strikingly, the conformational transition in grafted PEG2000 chains on PBS could not be easily described by the existing interpretive schemes. Plausibly, this transition becomes partially impaired due to interactions with PBS. Thus, saline has a significant effect on miscibility, phase, and conformational transitions in these PEG-grafted monolayers bearing C-16 aliphatic chains, which may have implications for understanding the behavior of PEG-grafted phospholipid surfaces in aqueous media of biological relevance.