The absorption spectrum of acridine orange in a monolayer or an LB membrane is studied using optical waveguide (OWG) spectrometry. Octadecyl acridine orange (C-18-AO) is incorporated into the stacked base pairs of octadecyl adenine (C-18-Ade) and octadecyl thymine (C-18-Thy), which are known to form a stable monolayer at air-water interfaces. Visible absorption spectra of the monolayer, which are difficult to obtain by conventional spectrometry, are easily generated by OWG spectrometry. A linear relation between the absorbance Of C-18-AO and the amount of monolayer deposited on the substrate was clearly discerned. This suggests that quantitative analysis of the monolayer can be performed by OWG spectrometry. Quantitative analysis can also be made of the structure of LB films. The absorbance at 500 nm, which is a characteristic of acridine orange, increased proportional to the number of layers on the substrate. The principal advantage of OWG spectrometry is in detecting the absorbance of molecules on any substrate. The absorption at 500 nm based on C-18-AO decreases exponentially with the gap between the LB film and the waveguide surface, because the intensity of the evanescent wave falls off exponentially with distance from the waveguide surface. The visible spectra were obtained under noncontact conditions when the gap between the waveguide and sample plate was less than 0.5 mum.