Three human pregnancy-associated plasma proteins (PAPPs), which were detected only during pregnancy with appropriately absorbed antisera to pregnancy plasma, were purified from third trimester plasma. The authors employed combinations of sequential DEAE-cellulose and hydroxylapatite chromatography; Sephadex G-100, G-200 and Sepharose 6B gel filtration; isoelectric focusing; and/or ammonium sulfate salting out. Each final product was shown to contain only one PAPP, to be immunologically free of most normal plasma proteins, and to have specific activities 100- to 145-fold greater than in the original pregnancy plasma. Rabbits immunized with these preparations responded with antisera which became mono-specific to their respective PAPP after appropriate absorption. PAPP-D, the one with low molecular weight (20,000), was shown immunologically to represent human placental lactogen. Another, PAPP-C, has been found to be identical to the pregnancy-specific β1-glycoprotein described by Bohn (designated by him as SP1 protein). The other, PAPP-A, of molecular weight 750,000, is distinct from the other two pregnancy-associated proteins of Bohn (designated SP2 and SP3), as well as the α2-pregnoglobulin of Berne. Since the latter and SP3 proved to be identical immunologically, and since it has been reported that SP3, Xh factor, pregnancy-associated globulin (PAG or Pal), and the pregnancyzone proteins are identical to each other, PAPP-A may represent a new pregnancy-specific protein. None of the PAPPs were detected in sera from 102 patients with various malignancies.