Purpose. A novel, non-reversible, aqueous-based lipidization strategy with palmitic acid as a model lipid was evaluated for conjugation with salmon calcitonin (sCT).Materials and Methods. A water-soluble epsilon-maleimido lysine derivative of palmitic acid was synthesized from reaction of palmitic acid N-succinimidyl ester and epsilon-maleimido lysine. The latter was generated from reaction of alpha-Boc-lysine and methylpyrrolecarboxylate, with subsequent deprotection of the Boc group. The palmitic derivative was further conjugated with sCT via a thio-ether bond to produce Mal-sCT in aqueous solution. The identity and purity of Mal-sCT was confirmed by Electrospray Ionisation Mass spectrometry (ESI-MS) and HPLC.Results. Yield of Mal-sCT was 83%. Dynamic light scattering and circular dichroism data suggested that Mal-sCT presented as a stable helical structure in aqueous solutions of varying polarity, with a propensity to aggregate at concentrations above 11 mu M. Cellular uptake of Mal-sCT was twice that of sCT in the Caco-2 cell model, and the conjugate was more resistant to liver enzyme degradation. Mal-sCT exhibited comparable hypocalcemic activity to sCT when administered subcutaneously in the rat model at sCT equivalent dose of 0.114 mg/kg. Peroral Mal-sCT, however, produced variability in therapeutic outcome. While four out of six rats did not respond following intragastric gavage with Mal-sCT, two rats showed significantly suppressed plasma calcium levels (similar to 60% of baseline) for up to 10 h.Conclusion. A novel non-reversible, water-soluble lipid conjugate of sCT was successfully synthesized that showed (1) different aggregation behavior and secondary structure, (2) improved enzymatic stability and cellular uptake, and (3) comparable hypocalcemic activity in vivo compared to sCT.