Lipoprotein-a [Lp(a)], one of the most atherogenic lipoproteins, is composed of a low-density lipoprotein (LDL) core in addition to an apo-a of variable size which is linked to apoB by a disulphide bridge. Lp(a) synthesized in vitro by incubation of recombinant apo-a (r-apo-a) with LDL is physico-chemically indistinguishable from native Lp(a). The synthesis of Lp(a) in vitro proceeds in two steps. In the first step, one of the unique kringle-IVs (K-IVs) in apo-a binds to a Lys residue of apoB; in the second step, Cys-4057 of K-IV type-9 (T-9) forms a disulphide bridge with Cys-3734 of LDL. Here we have produced r-apo-a with different combinations of unique K-IVs and shown that K-IV T-6 is required for the first step of Lp(a) assembly. For the second step not only is K-IV T-9 essential, but also the distance between T-6 and T-9 requires a length of two K-IVs. These findings give additional insight into the mode of Lp(a) assembly and are of relevance in the search for apo-a mutants influencing Lp(a) levels and for the development of Lp(a)-lowering medications.