This study was aimed at understanding the nature of conformational transition (s) that occur in poly (ethylene glycol) chains with molecular weight 5000 (PEG5000) grafted onto phospholipid monolayers. The study was performed with monolayers of PEG−phospholipid, DSPE−PEG5000, at the air/water interface and on solid substrates. Surface pressure and surface potential measurements together with ellipsometry and Brewster angle and atomic force microscopy were used to assess changes in the conformation and hydration of PEG5000 chains with increasing Peg grafting density. A comparative analysis of our experimental data suggests that neither the concept of the first-order pancake-to-brush conformational transition nor the model considering two subsequent transitions, namely, pancake-to-mushroom and mushroom-to-brush transitions, are adequate in describing conformational changes in the DSPE−PEG5000 monolayer at the air/water interface. At low grafting densities, PEG5000 chains did behave as grafted polymeric chains in a good solvent forming true 2D pancakes at the air/water interface. However, the conformation developed at high grafting densities was found to differ significantly from the PEG5000 brush in a good solvent. Its extension into the subphase was in far better agreement with the theoretical predictions for the height of PEG5000 brush in a theta solvent. This finding together with the decreasing absolute value of ψ0 potential of DSPE−PEG5000 monolayer implies that conformational transition (s) in PEG5000 are accompanied by changes in interactions of Peg monomers with water molecules and by a substantial dehydration of PEG5000 chains. Conformational changes in grafted PEG5000 chains at the air/water interface are therefore interpreted as a transition from pancake to poorly hydrated brush conformation.