Infrared spectroscopy (IR) and scanning tunneling microscopy (STM) have been used to analyse a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto alkane-derivatized surfaces, which were constructed using Langmuir-Blodgett (L-B) and self-assembly methods. The phospholipid used as the outer monolayer was 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG). The hydrocarbon-covered substrates which formed the inner half of the bilayer consisted of either (1) a perdeuterated stearic acid (d-SA) L-B monolayer on a Ge-attenuated total reflectance (ATR) element or (2) vapor-deposited Au-on-mica derivatized with a self-assembled monolayer of 1-octadecanethiol. Using ATR-IR spectroscopy of the DPPG/d-SA model system, the average conformation and orientation of the individual monolayer components in the bilayer membrane was determined. Conformational analysis of the C-H and C-D vibrational modes show that both halves of the hydrocarbon/deuterocarbon bilayer exist in an ordered conformational state. The average orientational distribution of the hydrocarbon chains in the DPPG monolayer was determined by an IR dichroic analysis to be approximately 10-15 degrees relative to the surface normal. In addition, the absence of a crystal-field splitting in the CH2 scissoring mode indicated a hexagonal subcell packing for the DPPG acyl chains in the upper half of this bilayer structure. Using STM, we have observed for the first time the formation of highly crystalline, nanometer-scale, hexagonal domains within the DPPG phospholipid monolayer. These hexagonal domains measured 7-10 nm in diameter with interior angles between adjacent domain boundaries of 119 +/- 2 degrees. Within these DPPG domains are observed molecularly resolved features of headgroup structure and packing. The unit-cell dimensions measured in these phospholipid domains correspond to a molecular area of 0.32 +/- 0.02 nm(2) for the DPPG headgroup. This result is in agreement with cross-sectional areas obtained from previous low-angle X-ray diffraction studies of the DPPG headgroup at low pH. Molecularly resolved defect structures within the phospholipid framework have also been observed in the STM images of this bilayer structure. The combination of IR spectroscopy and STM imaging indicates an extremely well-ordered, highly crystalline structure for this phospholipid membrane model.