A novel chemically modified insulin, -NB29-DHC-insulin (where DHC-insulin stands for dehydrocholyl-insulin), was prepared by covalent linkage of DHC (dehydrocholic acid) to the -amino group of LysB29 without any protecting agent and analysed by PAGE and reversed-phase HPLC. DHC-insulin was identified by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) coupled with dithiothreitol and trypsin treatment. The major product, mono-DHC-insulin, was -NB29-DHC-insulin. The biological activity of the modified insulin was evaluated by measuring the in vivo hypoglycaemic effect and in vitro tryptic degradation. Mono-DHC-insulin maintained the glucose-lowering effect as strongly as native insulin, showed a significantly slower disappearance due to an increase of its receptor-binding potency and retained approx. 40% of the reduced glucose relative to the undegraded sample digested for 6 h with trypsin in vitro. These results suggested that -NB29-DHC-insulin showed a longer duration of action compared with native insulin and also a protective effect from tryptic degradation.