Isotachophoresis of colored model proteins was carried out on polyacrylamide gel, using the stack of a multiphasic buffer system computed on the basis of the Jovin theory and operative at pH 10.4. The stack was elongated by milligram loads of various amino acids per analytical gel. The extent of the stack was determined by chemical localization of the leading and trailing constituents. The isotachophoretic nature of the stack was ascertained by determining the positions of all protein species under study as intermediate between the leading and trailing constituents. The shallow pH gradient across the extended stack was measured. Spacing of proteins in isotachophoresis is restricted, with regard to constituent multiplicity and constituent load, by the practical limitations of electrophoresis time and gel length. Therefore, spacing by a small number of constituents at relatively low loads was attempted. Amino acids were chosen as spacers because they can be selected conveniently, in radioactively labeled form, for the specific separation between pairs of proteins with particular mobilities. In the particular buffer system used, lysine, histidine, serine, and threonine were found to be effective spacers between bovine serum albumin (BSA) and hemoglobins, while no amino acids effectively spaced between hemoglobins A and S. It is concluded that specific spacing in isotachophoresis on polyacrylamide gel (ITPPA) can be useful in improving resolution. However, in practice, few spacers exist in the mobility range of proteins, the positions of which relative to specific proteins could be conveniently located on the basis of isotope analysis or other assays. Mixtures of multiple spacers with undefined mobihties appear applicable as "blind spacers" only at concentrations so low that their effectiveness is annulled.