We measured FT-IR spectra of intact Acholeplasma laidlawii cells grown at 37 °C on palmitic acid (C16:0) or on binary palmitic acid-d31/oleic acid (C16:0-d31/C18:1(9)) at an initial mole ratio of 2:3, which have been previously reported to produce significant fluctuations in CH 2 symmetric stretching ( ν sCH 2) and CD 2 asymmetric stretching ( ν aCD 2) frequencies (Biochim. Biophys. Acta 1279 (1996) 49). Time courses for acyl chain ν sCH 2 and ν aCD 2 frequencies determined from fourth derivative spectra are presented. Fluctuations were detected with the C16:0 enriched cells at temperatures above 40 °C as well as with the cells enriched in 2:3 C16:0-d 31/C18:1(9). These observations at temperatures above 40 °C for the C16:0 enriched cells were not in agreement with the conclusion in the previous work by Moore et al. Our results have suggested that the ∼2850 cm −1 ν sCH 2 band comprises two components arising from trans and gauche conformations, and that the fluctuations in ν sCH 2 frequency are caused by random temporal changes in the relative intensities of these two components.