Reversephase HPLC was utilized to study the synthesis, properties, and reactions of the neoglycopeptides formed by reductive lactosylation of the lysine amino groups of a derivative of the immunostimulatory thymic polypeptide thymosin (Y,. During the reaction of Nsformyldesacetylthymosin or, a 28-amino acid polypeptide which contains four lysines, with lactose and sodium cyanoborohydride, over 40 intermediates and products were separated by HPLC and 14 of these partially characterized. Increasing levels of lactosylation reduced the elution time of the modified thymosin a, derivatives in several HPLC buffer systems investigated. Furthermore, comparisons of mobilities of intact glucosyl-or lactosyl-N"-formyldesacetylthymosin (Y, showed that the main determinant of elution time was the total number of monosaccharide units per peptide molecule. HPLC analysis was also shown to be a useful tool for studying the susceptibility of neoglycopeptides to proteolytic degradation and will prove an aid to elucidation of the relative reaction rates of the individual thymosin OL, lysine residues. It is expected that the trends in peptide mobility described will generally apply to the behavior of naturally occurring glycopeptides and glycoproteins in reverse-phase HPLC.