The vitamin K-dependent clotting factors require posttranslational proteolytic processing before they are secreted by the liver into blood as mature zymogens. For most of these proteins, the sequences around the cleavage sites show a common motif (Arg-X-Lys/Arg-Arg) which define the substrate specificity for the endoprotease furin/PACE of the Golgi apparatus. In this paper, we present data which suggest that an additional Ca(++)-dependent endoprotease(s) is located in the endoplasmic reticulum, and may participate in processing of the two-chain vitamin K-dependent coagulation factor X. The single-chain 70 kDa factor X precursor in microsomes from rat liver was labeled by 14C-gamma-carboxylation and its conversion to a two-chain form followed in an incubation system with microsomal membrane fragments. A Ca(++)-dependent endoprotease(s) converted the factor X precursor to a two-chain form with a light-chain of 21 kDa. The endoprotease(s) showed little reactivity towards release of the factor X propeptide. The data provide new information about the endoprotease system in liver which participates in clotting factor proteolytic processing.