Calphobindin II, with Mr 73,000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental lambda gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2,361 nucleotides and a 64-nucleotide-long poly(A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.