Lipoprotein(a) (Lp(a)) consists of a low density lipoprotein particle in which apolipoprotein(a) (apo(a)), is disulfide linked to apoB. Lp(a) is produced by the liver, and high plasma levels represent an independent risk factor for cardiovascular diseases. However, pathways of production and metabolism of Lp(a) are poorly understood. We used primary cultures of baboon hepatocytes to analyze the steps involved in Lp(a) biogenesis. The results demonstrated that Lp(a) assembly was extracellular, since it was inhibited when anti-apo(a) antiserum was present in the culture medium. In addition, free apo(a) produced by hepatocytes could associate extracellularly with apoB in either very low density or low density lipoproteins. Lp(a) assembly required lysine-binding pockets in apo(a) kringles, as it was inhibited by the lysine analog, 6-amino hexanoic acid. A portion of apo(a) was also bound to the cell surface via its kringle domains and could be released into the medium by 6-amino hexanoic acid or proline. In add-back experiments, apo(a), but not Lp(a), bound to the cell surface. Addition of low density lipoprotein or very low density lipoprotein to hepatocyte cultures released apo(a) from the cell surface into the lipoprotein fraction of culture medium. We conclude that assembly of Lp(a) can occur at the cell surface. This represents one potential mechanism of Lp(a) production in vivo.