Atomic force microscopy (AFM) and FTIR spectroscopy techniques have been exploited to investigate the inverted hexagonal phase (H-II) of cardiolipin obtained by dehydration of a phospholipid water dispersion on a solid support. The characteristic cylinders of the H-II phase have been imaged by AFM and the effects of different preparation conditions (temperature and the presence of chemicals) on the structural parameters and on the presence of local nanoscale defects have been studied. It has been found that the measured repeat spacing of the H-II cylinders decreases upon increase of temperature and addition of pentachlorophenol (PCP), a chemical which is known to affect the structure and function of lipid bilayers. It has been shown that AFM can help in revealing some features of the mechanism of the inverted hexagonal phase formation, corroborating the results of a recent molecular dynamics study on the H-II phase formation from multilamellar phospholipid structures.