Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.