Reversible aqueous lipidization (REAL) at the interdisulfide bond has been shown to improve the deliverability of some peptide drugs. Recently, we developed a nonreversible aqueous lipidization method targeted at the interdisulfide bond of salmon calcitonin (sCT). The resultant derivative had comparable hypocalcemic activity to sCT after subcutaneous injection in rats, despite possessing significantly different biophysical properties. The purpose of this study was to conduct a comparative evaluation of the biophysical properties of the reversible aqueous-soluble lipidized sCT (REAL-sCT) and its corresponding nonreversible aqueous-soluble compound (Mal-sCT) with a view to correlate these properties to the bioactivities of the peptides. REAL-sCT and Mal-sCT were successfully synthesized, purified and identified. Both conjugates showed comparable retention times in a C-18 HPLC column, as well as robust helical structures and aggregation behavior in water, although REAL-sCT was shown by dynamic light scattering experiments to form larger aggregates than Mal-sCT in water. The larger particle size of REAL-sCT correlated with its stronger resistance to degradation by intestinal enzymes. Unlike Mal-sCT, REAL-sCTwas rapidly converted to sCT in liver juice; however, the regenerated sCT appeared to degrade at a slower rate than unmodifed sCT in the liver juice. Compared with sCT, REAL-sCT after subcutaneous injection as an aqueous solution at a dose of 0.15 mg/kg produced a prolonged hypocalcemic activity that lasted at least 24 h in the rat. Using a novel LC-MS/MS method that was developed for this study, we were able to show concomitant increases in REAL-sCT and sCT plasma concentrations with time, the latter prevailing at 10% the molar concentration of the former. In contrast, sCT was not present in the plasma following the subcutaneous injection of Mal-sCT, although a comparable hypocalcemic activity with shorter duration was observed. Oral administration of REAL-sCT and Mal-sCT as aqueous solutions at sCT equivalent dose of 5.0 mg/kg did not produce significant hypocalcemic activity. This study is the first thorough examination of the biophysical characteristics of the corresponding reversible and nonreversible aqueous-soluble lipidized peptide molecules. The results obtained should be useful for the development of the oral formulation of peptide and protein drugs.