A systematic investigation of the controversial association between indigo and palygorskite in Maya blue (MB), the famous Mesoamerican nano-hybrid pigment, is presented. It is based on vibrational spectroscopy up to near-infrared frequencies, as well as on X-ray diffraction and thermogravimetric analysis. The vibrational data point to a unique vibrational signature of the associated indigoid species over a broad range of experimental parameters (different octahedral composition of palygorskite, indigo concentration up to ca. 10 wt%, preparation temperature up to ca. 130 A degrees C). This signature of MB includes bands attributed to the fundamental, combination, and overtone modes of N-H groups and proves that the associated species is indigo and not dehydroindigo. The high-wavenumber position of the N-H modes suggests that indigo is interacting weakly with the host matrix. The association with palygorskite proceeds with a log(time) kinetic law, provided that the tunnels of the clay are free from zeolitic H(2)O. It is found that palygorskite with pre-dried tunnels forms MB even at ambient temperatures. On the contrary, palygorskite with pre-folded tunnels does not associate with indigo. These data are compatible with models involving the diffusion of indigo inside the tunnels of palygorskite. By filling the tunnels, indigo blocks their rehydration at ambient as well as their folding at higher temperatures. At ca. 220 A degrees C, indigo in MB assumes quantitatively a different bonding arrangement. This high-temperature structural transformation of MB is slowly reversible upon rehydration at ambient and its onset is observed at relatively low temperatures (a parts per thousand yen130 A degrees C). Thus, MB synthesized above ca. 130 A degrees C and not given sufficient time to rehydrate fully at ambient would still contain variable amounts of the high-temperature species, which may explain conflicting reports about the nature of the guest molecule.