Mixed distearoylphosphatidylethanol amine (DSPE) and dioleoylphosphatidylethanolamine (DOPE) monolayers and bilayers have been deposited on mica using the Langmuir-Blodgett (LB) technique, as a model system for biomembranes. Investigation with atomic force microscopy revealed phase-separation for both monolayers in air and bilayers in water in the form of microscopic DSPE domains embedded in a DOPE matrix. For the monolayers in air, the step height measured between the higher DSPE phase and the lower DOPE phase was larger than expected from the molecular lengths, and a significant contrast in adhesion and friction was observed despite identical lipid end groups. This unexpected behavior resulted primarily from a difference in the film mechanical properties, the DOPE phase being inelastically deformed by the probe. For the bilayers in water, similar trends were found in terms of height, adhesion, and friction, but an additional short-range repulsive hydration/steric force over the DSPE phase contributed to the observed differences.