The monolayer formation properties of cholesterol-based azobenzene amphiphiles with the natural (S)-C3 configuration (1(S)) and the inverted (R)-C3 configuration (1(R)) were examined at the air-water interface. The computational study reveals thas 1(S) adopts an extended conformation whereas 1(R) adopts an L-shaped bent conformation. 1(S) gave an expanded phase with A(0) (limiting area) = 0.60 nm(2) molecule(-1) and A(1) (lift-off area) = 0.64 nm(2) molecule(-1) whereas 1(R) gave a condensed phase with A(0) = 0.49 nm(2) molecule(-1) and A(1) = 0.54 nm(2) molecule(-1). Examination using reflectance spectroscopy established that 1(S) forms a monolayer with a J-aggregation mode (lambda(max) 407 nm) and with an increase in the compressibility it changes to an H-aggregation mode (lambda(max) 336 nm) whereas 1(R) forms a monolayer having a lambda(max) (362 nm) comparable with the monomeric absorption maximum, indicating that the electronic interaction among the azobenzene moieties is absent. The morphological changes in the monolayers were directly observed by an optical microscope and reasonably correlated with the spectroscopic changes. The results indicate that the absolute configuration at C3 has a crucial influence on the aggregation properties of cholesterol-based amphiphiles in a monolayer system.