The surface pressure-dependent conformational state of a monolayer film of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water (A/W) interface was studied using infrared external-reflection spectroscopy and two-dimensional infrared (2D IR) correlation analysis. When the IR spectra of the DPPC monolayer was collected using polarized IR radiation, a band splitting was observed in both the antisymmetric (νa) and symmetric (νs) methylene CH2 stretching modes that was not observed with unpolarized radiation. This band splitting was interpreted as being due to the presence of co-existing ordered and disordered conformational states, however, definitive identification of conformational sub-bands is problematic due to the low signal-to-noise inherent in the polarized IR spectra. To further investigate the spectral changes observed in the C-H region, 2D IR correlation analysis was applied to a set of pressure-dependent unpolarized IR spectra of the DPPC monolayer. When these unpolarized spectra were analyzed using 2D IR methods, the 2D asynchronous correlation spectrum of the DPPC monolayer clearly showed that cross peaks attributable to the νa and νs CH2 bands both split into two components, in agreement with the polarized IR monolayer spectra. Since band splitting in 2D IR spectra may be due to several causes, computer simulations were undertaken to help elucidate the exact cause of the observed splitting in the DPPC 2D asynchronous spectrum. Synthetic monolayer IR spectra were calculated for two limiting cases. The first was a frequency shifting model in which a single band underwent a simple frequency shift. The second limiting case was an overlapped peaks model in which an overall vibrational band was calculated as the sum of two individual sub-bands whose frequencies remained constant, but whose relative intensities changed through the simulated monolayer transition. The results of the computer simulations indicated that a simple frequency shift could be distinguished in the 2D asynchronous spectrum by the presence of a quartet of cross peaks, two with positive correlation intensities, and two with negative. In addition, a curved elongation of these cross peaks along the diagonal was associated with this frequency shift. In contrast, the 2D asynchronous spectrum for two overlapped peaks resulted in a correlation intensity cross peak doublet, one positive and one negative with no elongation along the diagonal. The experimentally measured 2D IR asynchronous correlation spectrum for the DPPC monolayer closely resembled the computer-simulated spectra for the overlapped peaks model. Therefore, the origin of the band splitting in the ν2 and νs CH2 bands in the 2D asynchronous spectrum is due to overlapping sub-bands that represent the ordered and disordered conformational states of the monolayer. Furthermore, these results also support the interpretation that the sub-bands observed in the polarized monolayer IR spectra are correlated with ordered and disordered monolayer states. © 2000 Elsevier Science B.V.