Conformational changes of alpha-mycolic acid (alpha-MA(MAI), isolated from Mycobacterium avium-M. intracellulare complex) in its monolayer at different surface pressures were investigated by using the Langmuir-Blodgett (LB) technique and atomic force microscopy (AFM). Three representative monolayers that correspond to the gas, liquid-expanded, and solid states were prepared and transferred onto freshly cleaved mica. The monolayer LB films of alpha-MA(MAI) were examined by AFM in an angstrom resolution in the thickness direction. The observation of topography and cross section of the monolayers proposed a series of schematic pictures for the lateral arrangement of the alkyl chains of alpha-MA(MAI) in the monolayer at different surface pressures. The proposed molecular conformations were consistent with the surface pressure-area isotherm. The results suggested that the alpha-MA(MAI) molecules partially took a flat-on stance in the gas-state monolayer and that the full extension of the longer alkyl chain of the molecule in the monolayer was possible only when the monolayer was highly compressed to the solid state.