Conformational changes of alpha- and keto-mycolic acid monolayers, which respond to monolayer compression, have been compared by use of the Langinuir-Blodgett (LB) technique and infrared reflection-absorption (RA) spectroscopy. Mycolic acids (MA) are major chemical components specifically found in cell envelopes of mycobacteria, and they are considered to largely influence the cell properties such as drug permeability. In our previous study, the longhydrocarbon chain in alpha-MA was found to have at least two distinct molecular conformations during the monolayer compression: fully extended and folded forms. In the present study, a surface pressure-area isotherm of keto-NU has suggested that keto-AM molecules have a different response of conformational changes to the monolayer compression from that of a-NU. The infrared RA spectra of the monolayer LB films were readily collected to monitor the conformational changes. The second-derivative analysis of the RA spectra clearly suggested that the long hydrocarbon chain in the keto-MA molecule remains folded even when a high surface pressure is applied to the monolayer, while the long chain in alpha-MA is unfolded. These results suggest that the small substituted keto group greatly influences the change of molecular conformation against the external pressure.