The interaction of recombinant apo(a) (r-apo(a)) with low-density lipoprotein (LDL) has been examined using ultracentrifugation and affinity chromatography. R-apo(a) forms a non-covalent complex with human LDL. This LDL-r-apo(a) complex, reconstituted Lp(a), r-Lp(a), which can be isolated by ultracentrifugation, has protease activity. The protease activity reached maximum at an equimolar ratio of r-apo(a) and LDL. Proline and epsilon aminocaproic acid (at a concentration of 50 mM) caused dissociation of r-Lp(a) and simultaneous loss of enzyme activity. Mouse LDL that did not form a complex with r-apo(a) did not activate the protease region of r-apo(a). Unlike plasma Lp(a), r-Lp(a) was dissociated during affinity chromatography on Lysine-Sepharose. This dissociation led to loss of enzyme activity. We conclude that the formation of a non-covalent complex between r-apo(a) and LDL leads to activation of the protease region of r-apo(a). The results suggest that non-covalent binding between r-apo(a) and LDL is a pre-requisite for the enzyme activity of the protease region of r-apo(a).