The acquired enamel pellicle is thought to be the result of a selective adsorption of salivary proteins and to be involved in the protection of the enamel surfaces. The chemical composition of the 2-h acquired enamel pellicle is fairly well established. However, the rate of formation and the amino acid composition of the initially formed enamel pellicle have been little investigated. The aim of this study was therefore to examine the rate of pellicle formation and the amino acid composition of the initially formed enamel pellicle. Samples of human enamel surfaces were carried in the mouth for various periods of time (2.5 min to 10 h). Rate of pellicle formation was indicated as a function of oral exposure time and the time necessary to remove the proteinaceous film from the surfaces by argon ion sputtering. The chemical composition of the initially acquired pellicle was examined by amino acid analyses of pellicle material collected in vivo from enamel surfaces 15 min and 1 h after pumicing, respectively. The pellicle reached an initial thickness in about 2–3 min, at which level it stayed for about 30 min. The thickness of the acquired pellicle then increased to about three times the initial thickness and stayed at that level for the rest of the experimental period (10 h). Amino acid analyses of pellicle material collected after 15 min and after 1 h were different in that the amino acid profiles of the 15-min pellicle only contained traces of proline and arginine. It may be argued that the pellicle formation proceeds in two stages owing to the adsorption of protein aggregates and that the chemical compositions of the pellicles of the two stages differ.