Grazing incidence X-ray diffraction (GIXD) measurements and crystallographic refinement calculations were used together to determine the molecular packing in pentacene monolayers on silicon oxide and investigate their relation with charge transport. The pentacene molecules in the first monolayer on silicon oxide were found to adopt a completely tilt-free herringbone motif, in comparison to the commonly observed thin-film phase or the bulk crystal. The theoretical charge-transport properties of the monolayer and bulk packing motifs were investigated on the basis of density functional theory calculations. The results offered an explanation for the high performance of pentacene TFTs relative to that of pentacene single crystals on the same substrate. Scattering from a smooth thin film in semi-kinematical approximation was also assumed for calculating the theoretical diffraction intensities.