Human milk bile-salt-stimulated lipase ensures efficient utilization of milk lipid in breast-fed infants. The N-terminal two-thirds of the peptide chain is highly conserved and shows striking similarities to typical esterases. In contrast, the remaining C-terminal part consists of a unique sequence of 16 proline-rich O-glycosylated repeats of 11 residues each. Recently we could show, using recombinant lipase variants, that neither these repeats nor the single N-linked sugar chain are essential for catalytic efficiency. In the present study, we report on the lack of importance of glycosylation and the unique repeats for other important functional properties, i.e. bile-salt activation, heparin binding, heat stability, stability at low pH and resistance to proteolytic inactivation. Compared to native enzyme, recombinant full-length lipase produced in two mammalian cell lines differed slightly in glycosylation pattern with no effects on the functional properties. Moreover, a variant lacking all repeats and the C-terminal tail following the last repeat exhibited the same functional characteristics as purified native milk enzyme. Thus, the structural basis for all the typical and functionally important properties reside in the N-terminal conserved part, in spite of the fact that none of these properties are shared by typical esterases. We could however, demonstrate that the C-terminal repeats are responsible for the unusual behaviour of the enzyme in size-exclusion chromatography, resulting in a considerably higher than expected apparent molecular mass.